Primate intra-acrosomal sperm antigen for use in a contraceptive vaccine

ABSTRACT

A substantially purified intra-acrosomal primate sperm antigen useful in a contraceptive vaccine is disclosed herein. The antigen remains associated with primate sperm after the acrosome reaction. In particular, it remains associated with the inner and outer acrosomal membranes. Modified antigens and fragments thereof prepared by protein modification techniques are also disclosed as well as methods for purifying and using the antigens. Also disclosed are monoclonal and polyclonal antibodies to the antigen and methods of making and using such antibodies. Methods of use include purification of the antigen or use in various diagnostic techniques. Also disclosed are cDNA, expression vectors, and transformed microorganisms that produce the antigen.

This application is a continuation of of application Ser. No. 08/292,045filed on Aug. 18, 1994, now U.S. Pat. No. 5,602,005 issued Feb. 11, 1997which is a continuation application of application Ser. No. 07/858,798filed on Mar. 27, 1992, abandoned, which is a continuation-in-partapplication of application Ser. No. 07/481,491, filed on Feb. 16, 1990,now U.S. Pat. No. 5,436,157 issued Jul. 25, 1995, which is acontinuation-in-part application of application Ser. No. 07/318,551,filed on Mar. 3, 1989, abandoned.

FIELD OF THE INVENTION

This invention relates to contraceptive vaccines. In particular, itrelates to a class of intra-acrosomal human and other primate spermantigens for use in contraceptive vaccines, a class of monoclonal andpolyclonal antibodies to the antigens, and related methods of making andusing the antigens and antibodies, including a cDNA expression systemfor the production of the antigens in vitro.

REFERENCES

Several publications are referenced herein by Arabic numerals withinbrackets or parentheses. Full citations for these references may befound at the end of the specification immediately proceeding the claims.The disclosures of these publications are hereby incorporated herein byreference in their entirety unless otherwise noted.

BACKGROUND OF THE INVENTION

Antibodies to sperm have been implicated in human infertility, and thedeliberate immunization of animals with sperm or mature testis extractshas resulted in a significant inhibition of fertility. Accordingly,researchers have actively pursued the study of sperm antigens in thehopes of identifying a germ cell specific antigen that can be used as animmunogen in a contraceptive vaccine. The approach of identifying gametespecific antigens has the advantage over other approaches, such as theHCG vaccine, of being a pre-fertilization vaccine--one which inducesimmunity which blocks fertilization as opposed to attacking the earlyembryo.

A safe and effective contraceptive vaccine would be a highly desirablemethod of birth control because a single injection or only a very fewinjections could provide antifertility activity in a human female forseveral years. However, until relatively recent biotechnologic andimmunologic advances, very few antigens suitable for such antifertilityvaccines had been identified and purified, especially in humans.Further, human proteins were significantly more difficult and expensiveto produce than most viral or bacterial proteins, and were not asimmunogenic. The emergence of hybridoma and recombinant DNA technologyhas provided the possibility of identifying germ cell specific antigensand mass producing the human form of such protein antigens for study andpotential use in new vaccines.

Anderson and Alexander, Fertility and Sterility, 40:557-571 (1983)discusses the general application of genetic engineering and monoclonalantibody technology to developing antifertility vaccines. It alsodiscusses some of the candidates for such vaccines, including the spermantigens protamine, lactate dehydrogenase-C₄ (LDH-C₄), RSA-1, acrosin,and hyaluronidase. The authors state that LDH-C₄ has been purified andamino acid sequence information is available. They further state thatmonoclonal antibodies (MABs) have been developed to it. The authors alsostate on page 561 that (1) sperm plasma membrane autoantigens providethe best targets for the effects of antifertility antibodies and (2)antigens bound to the inner acrosomal membrane, such as acrosin andhyaluronidase, appear to be poor candidates.

Over the last several years, many different monoclonal antibodies havebeen made to human and other animal sperm antigens. For example, Lee etal., Journal of Reproductive Immunology, 4:173-181 (1982), incorporatedherein by reference, discloses mouse MABs that react with antigenslocalized in the acrosomal region of human sperm. Such antigens areapparently on the surface of the sperm, and they have a molecular weightof about 10,000.

Another example is a mouse MAB, designated C11H, to an acrosomalantigen. See Kallajoki and Souminen, International Journal of Andrology,7:283-296 (1984), Kallajoki et al., International Journal of Andrology,9:181-194 (1986), and Salonen and Kallajoki, International Journal ofAndrology, 10:731-739 (1987), all of which are incorporated herein byreference. The 1984 paper discloses the preparation of C11H and that itrecognized an antigen of 50,000 molecular weight as well as othercomponents of 24,000-34,000 molecular weight. The antigen was found inthe sperm of humans and certain animals. The authors indicated that theybelieved the antigen to be acrosin, and they stated that they did notknow whether it was in the acrosome or within the acrosomal membranes.The 1986 paper provides further information about the antibody andantigen. The authors state that the antibody reacted with acrosin andfurther that acrosin is in the acrosomal matrix. They suggested thatacrosin is almost totally liberated during the acrosome reaction. Theyfurther state that it reacted against a 50 Kd antigen and several othersin the 24-34 Kd range. Finally, they state that the MAB can be used toscreen for acrosome-reacted sperm. The 1987 paper discloses experimentsin which C11H inhibited sperm penetration of zona-free hamster eggs.

Huneau et al., International Journal of Andrology 11:13-24 (1987),incorporated herein by reference, discloses a mouse MAB, designated a-HS1E.1, which reacts with human sperm in the equitorial region of theacrosomal membrane. FIG. 2 in the paper indicates that the MAB reactswith the outer acrosomal membrane. The corresponding antigen has amolecular weight equal to or greater than 53 Kd.

Such antibodies provided the possibility, at least in theory, ofidentifying, isolating, and characterizing gamete cell specific antigensthat might be useful in a contraceptive vaccine. However, the efforts todate have been disappointing.

Anderson et al., Journal of Reproductive Immunology, 10:231-257 (1987),incorporated herein by reference, discloses a multi-laboratory effortsponsored by the World Health Organization (WHO) to evaluate 66different mouse MABs that react with human sperm. Of the 66, only 3reacted with antigens that looked like good candidates for acontraceptive vaccine. One of these monoclonal antibodies, designatedMHS-10, showed strong human sperm and testicular germ cell reactivityand a lack of cross-reactivity with many other adult tissues. Theantibody inhibited sperm/egg binding in the hamster egg penetrationtest. The authors also stated that MHS-10 bound to a human sperm surfaceantigen and that it reacted with a family of antigens with molecularweight between 14,000 and 30,000.

The authors disclosed various difficulties in evaluating this MAB andthe other MABs. They concluded from their evaluation of all of theantibodies that the mouse monoclonal antibody approach is not efficientfor the identification of human reproductive tissue-specific antigensand further that the immunohistological data and the "surprisingcross-reactivity of most of! the MABs with non-reproductive tissues"underline the necessity for extensive immunohistologic testing of newMABs by qualified immunopathology groups.

At least one recent study continues to reflect the conventional wisdomthat an antifertility antigen should appear on the sperm surface.Primakoff et al., Nature, 355:543-546 (1988), incorporated herein byreference, reports an affinity-purified guinea pig sperm protein,designated PH-20, which was used as an immunogen to prevent conceptionin male and female guinea pigs. The antigen, which has a molecularweight of 64,000, is present on both the plasma membrane and inneracrosomal membrane of guinea pig sperm. In the last paragraph of thearticle, the authors state that the high contraceptive effectiveness ofthe antigen depends upon several specific properties, including itspresence on the sperm surface. They further state that a humanfunctional analog of PH-20 would be a candidate for an effectivecontraceptive immunogen.

Herr et al., Journal of Andrology, 9:42 (1988) is an abstract thatreports further data on the antigen identified by MHS-10. In particular,it discloses that the antigen is localized to acrosome-shaped structuresin the human sperm and that the peptide has 7 major isoforms with amolecular weight of 22-38 kD.

MSH-10 and its corresponding antigen, human acrosomal sperm antigen 10(SP-10), have now been substantially purified and characterized by theinventors. The inventors have surprisingly discovered a class ofintra-acrosomal human sperm antigens that, contrary to the conventionalwisdom, may have antifertility activity when used as an immunogen in acontraceptive vaccine for human females. The inventors have also foundthe SP-10 antigen in other primates. The inventors have isolated thecDNA coding for the SP-10 antigen, which permits the use of geneticengineering methods for making the antigens in a form useful as aimmunogen in a vaccine.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a substantially purifiedintra-acrosomal primate sperm antigen and immunogenic polypeptides foruse in a contraceptive vaccine. Another object of the invention is toprovide compositions for use as a contraceptive vaccine.

A further object of the invention is to provide methods for producingthe antigen.

It is a further object of the invention to provide monoclonal andpolyclonal antibodies that react with the intra-acrosomal primate spermantigen of the invention and methods for producing the antibodies.

Still another object of the invention is to provide a composition andmethod for detecting human sperm or isolating such sperm.

Still another object is to provide methods and compositions for thebiochemical, immunological, functional, or other investigationalanalysis of human sperm.

Still another object of the invention is to provide a method fordetecting immature germ cells in semen and the application of thismethod in assessing infertility.

Still another object is to provide DNA molecules and expression vectorsthat code for the antigen and polypeptides of the invention.

A further object of the present invention is to provide immunogenicpeptides of the antigen.

Yet another object of the invention is to provide transformedmicroorganisms that produce the antigen and the polypeptides.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be attained by means of theinstrumentalities and combinations particularly pointed out in theappended claims.

To achieve the objects and in accordance with the purpose of theinvention, as embodied and broadly described herein, a substantiallypurified intra-acrosomal primate sperm antigen that remains associatedwith the sperm after the acrosome reaction is disclosed herein.Preferably, following the acrosome reaction, the antigen remainsassociated with the sperm head, and most preferably it is associatedwith either the outer aspect of the inner acrosomal membrane or with theequatorial segment of the sperm. Preferably, the primate antigen is ahuman antigen.

An alternative embodiment of the invention is a substantially purepolypeptide that exhibits substantial homology to this antigen or hasbeen altered to provide a polypeptide having enhanced antifertilityimmunogenicity when compared to the unaltered polypeptide.

Also described herein is a method for producing the native antigen ofthe invention. Mature primate sperm, preferably human sperm, arehomogenized, and soluble proteins are extracted. The extract iscontacted with an immobilized monoclonal antibody that reacts with theantigen of the invention to form an immobilized complex of the antibodyand the antigen. The antigen is then separated from monoclonal antibodyto be recovered in substantially purified form.

Also described herein is an alternative method for producing the nativeantigen from primate sperm, preferably human sperm. Ejaculated sperm arehomogenized and the soluble proteins are separated by reverse phase highpressure liquid chromatography. SP-10 peptides are then further purifiedby preparative SDS-PAGE.

The invention also provides a monoclonal antibody to the intra-acrosomalprimate sperm antigen. The antibody lacks cross-reactivity with asubstantially all primate somatic tissues and inhibits sperm-egginteractions in the hamster egg penetration test. Preferably, themonoclonal antibody reacts with the human sperm antigen.

The monoclonal antibody is produced by immunizing a mammal withacrosome-reacted primate sperm or the supernatant obtained fromcentrifuging acrosome-reacted primate sperm. Preferably, human sperm isused. The antibody-producing cells from the mammal are obtained andfused with tumor cells to produce hybridomas. The hybridomas arescreened with acrosome-reacted sperm or the supernatant obtained fromcentrifuging such sperm in order to identify hybridomas that produce theantibody reactive with the intra-acrosomal sperm antigen. The antibodyis then recovered from the identified hybridomas.

In addition to purifying the antigen of the invention, the monoclonalantibodies disclosed herein are useful for detecting or isolating spermthat have undergone the acrosome reaction. The antibody is contactedwith a sample of sperm for a time and under conditions sufficient forthe antibody and any acrosome-reacted sperm to form an antigen-antibodycomplex. The complexes are then detected or removed from the sample. Inthe latter case, the sperm cells are then separated from the complex andrecovered as an isolate.

In an alternative and preferred embodiment, the antigen of the inventionis produced by culturing host cells transformed by an expression vectorthat directs the expression of the antigen in the transformedmicroorganism. The expression vector comprises a recombinant DNAsequence containing a cDNA sequence that codes for the antigen operablylinked to appropriate regulatory control nucleic acid sequences.

The immunogenic polypeptides of the invention are similarly produced.Preferably, such polypeptides are the 265 amino acid protein designatedhuman SP-10 or its 246 amino acid variant. Alternatively, thepolypeptide is the 285 amino acid baboon or monkey SP-10 protein or its251 amino acid variant. The invention further provides immunogenicfragments of these proteins. Preferably, the fragment contains theepitope recognized by monoclonal antibody designated MHS-10. Mostpreferably, the fragment comprises the carboxyl terminus of therespective proteins.

The accompanying figures, which are incorporated in and constitute apart of the specification, illustrate an embodiment of the inventionand, together with the description, serve to explain the principles ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Immunohistochemical localization of SP-10 within seminiferoustubules of the human testis. FIG. 1A. Cross sections of seminiferoustubules reacted with the MHS-10 monoclonal antibody (1:1000) demonstratedark reaction product in the adluminal compartment. X 180. FIG. 1B. Athigher magnification, both crescent shaped and smaller granular reactionproduct (arrowheads) are observed in cohorts of similar stage germ cellswithin a single seminiferous tubule. X 720. FIG. 1C. Tissue sectiontreated with the control murine IgG₁ shows no staining. X 180.

FIG. 2. Immunofluorescent light micrographs localizing SP-10 inejaculated human sperm. FIG. 2A. A combination phase contrast andfluorescent image demonstrates cap-shaped fluorescence over the anteriorportion of the sperm head. X 2870. FIG. 2B. Sperm following artificialinduction of the acrosome reaction with the calcium ionophore A23187. Inthe experiment from which the above photo was taken, 47.5% of spermshowed full fluorescent caps, 20.3% faint fluorescent caps (curvedarrow), 22.4 % equatorial bars (straight arrow), and 9.9% of the spermwere unstained. X 3350.

FIG. 3. Electron micrograph of human sperm head following reaction withmonoclonal antibody MHS-10 and Protein-A gold. Gold particles areobserved over the acrosomal compartment. In regions where the acrosomewas sectioned obliquely as at the sperm apex, the gold particles followa bilaminar distribution. Arrowheads indicate location of acrosomalmembranes which are electron lucent in this unosmicated material. X98,300.

FIG. 4. One dimensional SDS-PAGE gel (10% acrylamide) electroblotted tonitrocellulose and stained with amido black (A) and identicalnitrocellulose sheet reacted with the MHS-10 mAb (B) or control IgG₁(C). Sperm extracts from 6 donors (1-6) contained B-mercaptoethanol(lanes marked R=reduced) or lacked this agent (nonreduced=N). 25 ugprotein was run per lane. The pattern of SP-10 immunoreactive peptidesis identical both between persons and in reduced and non-reducedextracts.

FIG. 5. Silver stained two dimensional gel (A) and immunoblot (B) usingMHS-10 MAB on proteins extracted from human sperm. A one dimensionallane showing the silver stain and immunoblot pattern of the spermextract lie at the right of each figure. Molecular weights (MW) andisoelectric points (pI) are indicated on the right and bottom margins,respectively. Arrows on the silver stain above indicate the location ofSP-10 proteins (AP) at 34 and 30 kDa which may be compared to bands andspots of similar mass on the immunoblot below. 2-D and 1-D gels wereloaded with 75 and 15 ug of sperm protein, respectively. ImmunoreactiveSP-10 peptides from 24-34 kd have a pI of 4.9, the 18 kd spots range inpI from 5.1-5.4.

FIG. 6. One dimensional SDS PAGE (16 cm gels) electroblots stained withAmido black (A) and with MHS-10 antibody (B). Lanes 1A & B contained 20ug human sperm proteins; lanes 2, 80 ug boar sperm proteins; lanes 3, 20ug purified boar proacrosin; lanes 4, 15 ug purified boar sperminogen.Monoclonal antibody MHS-10 (1/1000) recognized several peptides ofsimilar mass as human sperm SP-10 peptides within the boar spermhomogenate (lane 2B) but did not cross react with purified boar acrosin(lane 3B) or sperminogen (lane 4B). Control lanes 1-4C were reacted withanother IgG1 monoclonal antibody ascites.

FIG. 7. Immunoblot (minigels) of human (H), bull (B), rat (Rt) andrabbit (Rb) sperm extracted with 1% SDS. Each lane was loaded with 3 ugof protein which was separated by SDS-PAGE and transferred tonitrocellulose. Lanes were stained with Amido Black (lanes A), a 1:2000dilution of MHS-10 Mab ascites (lanes B), or a 1:2000 dilution of nullascites (lanes C). Lanes incubated with ascites were subsequentlyincubated with HRP-labelled goat anti-mouse IgG secondary antibodyfollowed by 0.05% DAB and hydrogen peroxide. The left lane containedmolecular weight standards of the indicated molecular weights.

FIG. 8. Immunoblot of human (Hs), Papio cynocephalus (Pc), Macacamulatta (Mm), and Macaca fascicularis (Mf) sperm extracted with 1% SDS.Each lane was loaded with 10 ug of protein which was separated bySDS-PAGE and transferred to nitrocellulose. Lanes were stained withAmido Black, a 1:2000 dilution of MHS-10 ascites, or a 1:2000 dilutionof null ascites as indicated. Lanes incubated with ascites weresubsequently incubated with HRP-labelled goat anti-mouse IgG secondaryantibody followed by 0.05% DAB and hydrogen peroxide.

FIG. 9. Northern blot of poly A+ RNA isolated from testes of human,baboon (Papio papio, Papio cynocephalus anubis), rhesus (Macacafascicularis), dog, and cat as well as human placenta and liver. Theblot was hybridized with a p³² labelled probe spanning 634 bp of theopen reading frame for human SP-10. A 1.35 kb mRNA is observed in lanescontaining human, baboon, and rhesus poly A+ testis RNA.

FIG. 10. Northern blot analysis of poly(A)+ RNA from human testes,liver, and placenta. One ug of testes poly(A)+ RNA, and 2 ug of liverand placental poly(A)+ RNAs were electrophoresed on a 1% formaldehydeagarose gel, transferred to Biotrace membrane and probed with a nicktranslated 634 bp SP-10-5 fragment. The SP-10 mRNA is approximately 1.35kb in length.

FIG. 11. FIGS. 11A & B. Complete nucleotide and predicted proteinsequences derived from overlapping SP-10-5 and SP-10-10 cDNAs. Thesingle letter amino acid code for the protein sequence is indicatedbelow the nucleotide sequence. The top line in each pair of sequenceswas derived from the SP-10-5 cDNA and the bottom line from the SP-10-10cDNA as indicated. The numbering to the right indicates the nucleotideand amino acid positions. The solid line in the SP-10-10 sequencespanning nucleotides 554-610 represents the putative alternativelyspliced region of SP-10-10. Repeated motifs one, two, and three aredesignated by single, double, and triple underlined sequencesrespectively. Sites of potential N-linked glycosylation are denoted bythe symbol -cho-, and sites of potential O-linked glycosylation areunderscored with the symbol (+++). The 5' consensus nucleotide sequenceflanking eukaryotic ATG start codons is underscored with the symbol ( ),a poly A addition signal is underscored with the symbol ( ), and a mRNAconsensus degradation sequence is underscored with the symbol (***). Thetwo in-frame termination codons 5' of the ATG are designated by TER. Aninternal EcoR1 site is indicated at the arrowhead. FIG. 11C.Hydrophobicity plot generated from the deduced SP-10-5 amino acidsequence. Hydrophobic residues lie above the center line and hydrophilicresidues lie below the line. The SP-10-5, SP-10-10, and SP-10-214 cDNAsare indicated below the the plot. The internal EcoR1 site at bp 695 isindicated.

FIG. 12. Western blot analysis of human sperm extracts using themonoclonal antibody MHS-10 and the SP-10 polyclonal antiserum generatedtoward recombinant fusion protein, pWRSP-210. MHS-10 and the polyclonalantiserum recognized an identical set of SP-10 polypeptides in spermextracts. Human sperm extracts were prepared and subjected to SDS PAGE,blotted, and incubated with the monoclonal and polyclonal antibodies asdescribed in Materials and Methods. Amido black staining of theelectrophoresed sperm extracts, lane 1. Extracts incubated with MHS-10,lane 2, or null ascities lane 3. Extracts incubated with the SP-10polyclonal antiserum, lane 4, or preimmune serum, lane 5.

FIG. 13. Immunofluorescent staining of human sperm using MHS-10 and theSP-10 polyclonal antisera. Both the MHS-10 monoclonal antibody and thepolyclonal antiserum to recombinant fusion protein pWRSP-210 react withthe acrosomal cap. Sperm incubated with SP-10 polyclonal antiserum,x1200 (A), preimmune sera, x1200 (B), MHS-10 monoclonal, x1775 (C), ornull ascities, x1775 (D).

FIG. 14. FIG. 14A. Cryostat section of human testis showingimmunohistochemical spermatids and mature sperm (top right-hand corner)(MHS-10, SBP, hematoxylin x 964). FIG. 14B. Early stage spermatid insemen prior to acrosome formation. Note lack of immunostaining withMHS-10 (MHS-10, SPB, hematoxylin x1600). FIG. 14C. Golgi phase spermatidin semen smear. Note the oval shaped immunostaining acrosomal granuleadjacent to the nucleus (arrow) (MHS-10, SPB hematoxylin, x2800). FIG.14D. Late Golgi phase spermatid in semen smear showing immunostainedacrosomal granule (arrow) and incomplete flagellum (MHS-10, SPB,hematoxylin x2600). FIG. 14E. Early cap phase spermatid in semen smear.Note uncondensed nucleus with immunohistostaining acrosome lyingproximal to implantation fossa (MHS-10, SBp hematoxylin x2205). FIG.14F. Mature sperm in semen smear showing a complete immunohistostainingacrosome (MHS-10, SHP, hematoxylin x194B).

FIG. 15. FIG. 15A. Immature germ cell in semen smear showing two nucleiwithin the same cytoplasm MHS-10, SBP, hematoxylin x2145). FIG. 15B.Spermatid in semen with two immunohistostained acrosomal granules withinthe same cytoplasm MHS-10, SBP, hematoxylin x2293). FIG. 15C.Biflagellated sperm with two condensed nuclei (arrows) and completedacrosomes within the same cytoplasm. Note too, the sperm with a largeuncondensed nucleus (lower left) and that with an abnormally largeacrosome (lower right). (MHS-10, SBP, hematoxylin x1583). FIG. 15D.Conjointed spermatids in semen smear displaying asynchronousdevelopment. Arrows point to acrosomal vesicles staged at Golgi phase(lower left hand corner) and to cap phase of formation, respectively(MHS-10, SBP, hematoxylin x1500). FIG. 15E. conjoined spermatids insemen smear (MHS-10, SBP, hematoxyline x1854). FIG. 15F. Biflagellatedspermatids in semen smear showing immunoreactive cap phase acrosome anduncondensed nuclei (MHS-10, SBP, hematoxylin x1672). FIG. 15G. Spermwith uncondensed nuclei and microacrosome in semen smear. (MHS-10, SBP,hematoxylin x1967). FIG. 15H. Cap phase spermatid lacking flagellum insemen smear (MHS-10, SBP, hematoxylin x2754). FIG. 15I. Pleomorphicfragments of sperm heads in semen showing immunohistostaining material(MHS-10, SBP, hematoxylin x2149). FIG. 15J. Spermatid in semen smearshowing a peripheral cuff of immunoreactive material (MHS-10, SBP,hematoxylin x2754). FIG. 15k. Spermatid in semen smear showing aperipheral cuff or immunoreactive material (MHS-10, SBP, hematoxylinx2368). FIG. 15L. Leukocyte in semen smear stained with anti-HLe-1. Notelack of immunohistostaining of spermatids and mature sperm in the samefield, (anti HL3-1, SBP, hematoxylin x845).

FIG. 16. Complete nucleotide sequences derived from overlapping SP-10cDNAs of baboon, macaque, and human. The cDNA sequences of macaque andhuman SP-10 are compared to the cDNA sequence of baboon SP-10. Thenumbering to the right indicates the nucleotide positions of the baboonand macaque cDNas. Matching nucleotides are denoted by an asterisk (*).Areas lacking comparable sequence are denoted by dashes (-----). Thetranslational start and termination codons are boxed and overscored withthe words "start" and "term", respectively. Nucleotides contained withinthe alternatively spliced introns are shaded. The first and last 3nucleotides, GTG and CAG, respectively, within the shaded region areunderlined denoting the primate consensus splice nucleotides. The 5'consensus sequence flanking the ATG start codon is underscored by thesymbol (+++++); the mRNA degradation consensus sequence is underscoredby the symbol ( ); the polyadenylation consensus sequence is underscoredby the symbol ( ). The major transcriptional start site is overscored byan arrow ( ) at nucleotide 4.

FIG. 17. Primer extension analysis of baboon and macaque SP-10 mRNAs.Both baboon (B) and macaque (M) have major extension products atnucleotide 4 of the cDNA sequences and minor products 2, 28, and 33nucleotides upstream from nucleotide 4. A synthetic oligonucleotidecomplementary to residues 75-94 in both species 5' d(GGGGATCCATTAGTAAGAGAAACATGTTCAT)! was used to generate the extensionproducts and the sequence ladder as described in Materials and Methods.The 1.2 Kb baboon SP-10 cDNA in pBluescript was used as the template forthe sequence ladder.

FIG. 18. PCR amplification of the open reading frame of baboon (B) andmacaque (M) SP-10. The PCR products have been separated in a 5%polyacrylamide gel and stained with ethidium bromide as described inMaterials and Methods. The upper band in baboon and macaque migrates at850 bp and the lower band migrates at 750 bp. This corresponds preciselyto the open reading frames of the 1.2 kB and 1.1 kB SP-10 cDNAs,respectively.

FIG. 19. Deduced amino acid sequence of baboon (B), macaque (M), andhuman (H) SP-10, and mouse (R) MSA-63. The deduced amino acid sequenceof macaque and human SP-10 and MSA-63 are compared to the deduced aminoacid sequence of baboon SP-10. The numbering to the right indicates thepositions of the amino acids. The hydrophobic leader sequence containsthe N-terminal 18 residues and is overscored by "sig. seq.". Exactmatches are denoted by an asterisk (*). Regions lacking comparablesequence are denoted by dashes (-----). Conserved cysteine residues aredenoted by downward-pointing triangles. The middle 50% of the sequencecontains the repeat motifs. The pentapeptide repeats are overscored fromabove: (S, E, H, G/A, A)→; (S/L, G, E, H, A, L)→; (S/V, G, E, Q,P/S/A)→. The 3 larger 25 amino acid repeat motifs are labeled above thepentapeptide symbols , , . The conserved N-linked glycosylation sitesare overscored by three downward-pointing arrows . Amino acids includedwithin the shaded region are encoded for by the alternatively splicedintrons. Alternative splicing results in SP-10 proteins with internaldeletions of 34 residues (baboon and macaque) and 19 residues (human).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the followingexamples, serve to explain the principles of the invention.

The invention relates to an intra-acrosomal human sperm antigen thatremains associated with human sperm after the acrosome reaction. Priorto the acrosome reaction, the antigen is observed within the acrosome.It is possible that the antigen is soluble in the acrosome matrix priorto the acrosome reaction. After the acrosome reaction, it remainsdisplayed on the sperm head. In a preferred embodiment, the antigen isassociated in intact, non-acrosome-reacted sperm with the outer aspect(face or side) of the inner acrosomal membrane and the inner aspect ofthe outer acrosomal membrane, and it remains associated with thosemembranes after the acrosome reaction. Most preferably, the antigen isretained in association with the inner acrosomal membranes and theequatorial segment in acrosome-reacted human sperm. As used herein, theterm "associated with" or variations thereof means bound with ahydrophobic tail inserted into the membrane or loosely bound byelectrostatic interactions and includes being unbound in the acrosomematrix prior to the acrosome reaction.

The antigen of the invention is testis specific and conserved in thehuman population. It appears to be a differentiation antigen that arisesduring spermatogenesis. It appears to be located in the acrosomal matrixof immature human sperm prior to the acrosome reaction.

This antigen has also been found in the sperm of non-human primates.Therefore, in its broadest sense, the antigen of the invention is anintra-acrosomal primate sperm antigen that remains associated with theprimate sperm after the acrosome reaction. As used herein, the term"primate" means the order of mammals comprising humans, apes, monkeys(both New and Old World), and prosimians, such as lemurs and tarsiers.

Preferably, the antigen is substantially purified. The terms"substantially pure" and "substantially purified" and variationsthereof, when used to refer to the antigen disclosed herein, shall meanthe antigen substantially free of proteins or polypeptides that are notthe intra-acrosomal primate sperm antigen. In the context of thepreferred antigen, substantially pure means that, when the purifiedantigen is sequenced by amino terminal amino acid sequencing, theresultant sequence compares with the deduced amino acid sequenceobtained from the open reading frame of the cDNAs. The substantiallypure antigen of the present invention is at least 90% pure by weight,preferably at least 95% pure by weight, and most preferably at least 98%pure by weight. That is, the substantially pure antigen of the inventioncontains no more than 10%, preferably no more than 5%, and mostpreferably no more than 2% by weight of proteins or polypeptides thatare not the antigen. The purity was determined by densitometric scanningof SDS-PAGE gels stained with Amido Black which contained the purifiedSP-10 antigen and by amino acid sequencing the NH₂ terminus of thepurified protein.

In a particularly preferred embodiment, the human antigen issubstantially purified and comprises a family of proteins orpolypeptides having a molecular weight from about 18 to about 34kilodaltons as determined by sodium dodesylsulfate (SDS) polyacrylamidegel electrophoresis. Immunoreactive peptides with molecular weights from24-34 kDa had an isoelectric point of approximately 4.9, whileimmunoreactive peptides in the 18 kDa range had pIs of 5.1-5.4, asdetermined by isoelectric focusing. The immunoreactive peptides appearto be single-chained, since a reduction of the disulfide bonds did notalter the apparent molecular weights. Most preferably, the antigen isone of these polypeptides.

In a particularly preferred embodiment, the primate antigen reacts withmonoclonal antibodies produced by the cell line designated ATCC HB 10039on deposit at the American Type Culture Collection, Rockville, Md.U.S.A. or mutants or variants thereof.

The antigen of the invention is obtained in substantially purified formby known protein extraction techniques that have been modified inaccordance with the discoveries and teachings described herein. Maturehuman or other primate sperm are collected and homogenized. The solubleproteins, including the antigen, are extracted from the homogenate byknown protein extraction techniques. The extract is then brought intocontact with a monoclonal antibody reactive with the antigen.Preferably, the monoclonal antibody is produced by the cell linedesignated ATCC HB 10039. The antibody and antigen react to form acomplex. Generally, the monoclonal antibody is immobilized, such as byconjugation to a solid substrate, so that the antigen may be removedfrom the extract. Preferably, the solid substrate, which contains theantibody-antigen complex, is then washed to remove other proteins andcontaminants. The antigen is then separated from the monoclonal antibodyby known techniques and recovered in substantially purified form. In analternative embodiment, a polyclonal antibody reactive with the antigenmay be used to purify the antigen.

In a preferred embodiment, the antigen is purified according to thetechnique disclosed in Isojima et al., Clin. Exp. Immunol., 49:449-456(1982) and Isojima et al., Immunological Approaches to Conception andPromotion of Fertility (Talwar Ed.), 323-333 (Plenum Publishing 1986),both of which are incorporated herein by reference. The homogenizedsperm extract is run through an immunoaffinity chromatography columnthat contains the monoclonal antibody immobilized upon a solid support,such as Sepharose 4B. The antigen is then eluted from the column bylowering the pH.

In an alternative embodiment, the extract can be run through a reversephase high pressure liquid chromatography (HPLC) column. The fractionsthat elute from the column are recovered. The protein components of thevarious fractions are separated by two dimensional gel electrophoresis.The component that contains the antigen is identified by reacting theblots on the gel with the monoclonal antibody and determining, byimmunochemical techniques, which component contains the antigen. Theantigen may then be recovered in substantially purified form by knowntechniques. The method has been verified by microsequencing the aminotermini of the purified peptides, and the amino acid sequences have beenshown to overlap with the amino acid sequences deduced from genecloning, thus confirming the usefulness of the method.

The substantially purified antigen of the invention may be furtherpurified by various protein purification techniques. The proteinpurification techniques include those identified and described in U.S.Pat. No. 4,446,122 issued May 1, 1984 to Chu et al., which isincorporated herein by reference. Preferably, the antigen is purified bypreparative electrophoresis or affinity purification.

Most preferably, the purification is accomplished by affinitychromatography followed by reverse phase HPLC and preparative SDS-PAGE.This technique is particularly useful for separating the particularpolymorphic polypeptides that appear to comprise primate SP-10.

In an alternative embodiment, the antigen of the invention may beisolated and purified from human sperm by general techniques well-knownin the art, modified and applied in accordance with the discoveries andteachings described herein. Such techniques include electrophoresis,centrifugation, gel filtration, precipitation, dialysis, chromatography(including ion exchange chromatography, affinity chromatography,immunoadsorbent affinity chromatography, reverse-phase high performanceliquid chromatography, and gel permeation high performance liquidchromatography), isoelectric focusing, and variations and combinationsthereof.

One or more of these techniques are employed sequentially in a proceduredesigned to separate molecules according to their physical and chemicalcharacteristics. These characteristics include the hydrophobicity,charge, binding capability, and the molecular weight of the antigen. Thevarious fractions of materials obtained after each technique are testedfor the ability to react with monoclonal antibody MHS-10, produced byATCC HB 10039. Those fractions showing such activity are then subjectedto the next technique in the sequential procedure, and the new fractionsare tested again. The process is repeated until only one fractionreactive with MHS-10 remains and that fraction produces only a singleband when subjected to polyacrylamide gel electrophoresis.

The antigen of the invention may be modified by known proteinmodification techniques. These include the techniques discloses in U.S.Pat. No. 4,302,386 issued Nov. 24, 1981 to Stevens and U.S. Pat. No.4,526,716 issued Jul. 2, 1985 to Stevens, both of which are incorporatedherein by reference. Such modifications may enhance the immunogenicityor antifertility activity of the antigen, or they may have no affect onsuch activity. For example, a few amino acid residues may be changed orremoved. Alternatively, the antigen of the invention may contain one ormore amino acid sequences that are not necessary to its immunogenicityor antifertility activity. It may be the case, for example, that onlythe amino acid sequences of a particular epitope of the antigen will benecessary for immunogenic activity. Unwanted sequences can be removed bytechniques well-known in the art. For example, unwanted amino acidsequences can be removed via limited proteolytic digestion using enzymessuch as trypsin or papain or related proteolytic enzymes. Alternatively,polypeptides corresponding to various immunogenic epitopes of SP-10 maybe chemically synthesized by methods well known in the art. Theseinclude the methods disclosed in U.S. Pat. No. 4,290,944 issued Sep. 22,1981 to Goldberg, incorporated herein by reference.

Thus, the antigen of the invention includes a class of modifiedpolypeptides, including synthetically derived polypeptides or fragmentsof the antigen, having common elements of origin, structure, andmechanism of action, such as antifertility effect, that are within thescope of the present invention because they can be prepared by personsskilled in the art, once given the teachings of the present invention.This includes any polypeptide derived from the deduced amino acidsequence of FIGS. 11A and B or FIG. 19, including fragments and variantsof the sequence, that is immunogenic and has an antifertility effectwhen injected into a mammal. For example, we have shown that one peptidefragment of SP-10 as shown in FIGS. 11A & B containing 71 amino acids(amino acids 143-213 in FIGS. 11A & B) reacts with the MHS-10 monoclonalantibody. SP-10 may contain other epitopes that react with MHS-10, whichcan be determined by persons skilled in the art. Accordingly, suchpolypeptides or peptide fragments are within the scope of the invention.Moreover, since persons skilled in the art can make modifications to orderivatives of such epitopes, such modifications or derivatives arewithin the scope of the invention, provided that they are immunogenicand have an antifertility or contraceptive effect in humans or otherprimates or other mammals.

The invention further comprises one or more substantially purepolypeptides that exhibit substantial homology to the antigen of theinvention or any of the polymorphic polypeptides that comprise it.Preferably, such polypeptide is at least 85% homologous to thereferenced polypeptide.

The monoclonal antibody used to identify, characterize, and purify theintra-acrosomal antigen is within the scope of the invention. It reactswith a substantially purified intra-acrosomal human or other primatesperm antigen that remains associated with the sperm after the acrosomereaction. Preferably, the antibody reacts with a human sperm antigenlocated in the acrosomal matrix of mature human permeabilized spermprior to the acrosome reaction and found in association with the inneracrosomal membrane or equatorial segment after the acrosomal reaction.

The particularly preferred monoclonal antibody of the invention lackscross-reactivity with substantially all human somatic tissues. Inaddition, the antibody inhibits sperm-egg interactions in the hamsteregg penetration test. These characteristics demonstrate reasonablyconclusively that the antigen of the invention or the active partsthereof can be expected to have antifertility activity after beinginjected into a human female.

In a particularly preferred embodiment, the monoclonal antibody of thepresent invention has the characteristics of the mouse monoclonalantibody produced by the hybridoma cell line ATCC HB 10039 or mutants orvariants thereof. ATCC HB 10039 is a biologically pure culture availablefrom the permanent collection of the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. USA, 20852 and wasdeposited there under the Budapest Treaty on Feb. 28, 1989. Theimmunoglobulins produced by this hybridoma are of the IgG1 isotype asdemonstrated by enzyme-linked immunosorbent assay employing isotypespecific reagents.

The monoclonal antibody of the invention is prepared by a modificationof the known techniques for the preparation of monoclonal antibodies andhybridomas. The modification reflects the discovery by the inventorsthat the antifertility antigen of the invention resides in the acrosomalmatrix prior to the acrosome reaction but it is then found inassociation with the inner and outer acrosomal membranes after suchreaction. This knowledge permits the modification of the conventionaltechnique to reproducibly provide monoclonal antibodies to antifertilityantigens found in association with the inner and outer acrosomalmembranes after the acrosome reaction.

Accordingly, the host animal is immunized with either acrosome-reactedhuman sperm or the supernatant obtained from centrifugingacrosome-reacted human sperm. In the first case, the sperm antigen isthen on the surface of the sperm, since it remains associated with theinner acrosomal membrane. In the second case, the supernatant willcontain high concentrations of the antigen, since it contains theremains of the outer acrosomal membrane, which also contains theantigen. The supernatant may also contain some of the antigen free insolution, since there is an indication that the antigen may also be freein solution in the acrosomal matrix prior to the acrosome reaction. Inboth instances, the material injected into the host contains an enrichedconcentration of the immunogens of interest. Thus, a larger fraction ofthe antibody-producing cells would be expected to produce the monoclonalantibodies of the invention.

Any host that produces antibodies may be used. Conventionally usedanimals include rabbits or rodents, such as rats or mice. Mice arepreferred for the present invention.

Once the animal has been immunized and sufficient time has passed for itto begin producing antibodies, the antibody-producing cells arerecovered. Although any antibody-producing cells may be used, Blymphocytes obtained from the animal's spleen are preferred.

The antibody-producing cells are fused with tumor cells to producehybridomas. As used herein, the term "tumor cell" includes any cell thatis capable of fusing with an antibody-producing cell to produce a hybrid"immortal" cell; i.e., one which is capable of continuous grown invitro. Preferred tumor cells are antibody-producing cells that have beentransformed and which have lost their ability to produce immunoglobulin.Such cells include rat myeloma cells and mouse plasmacytoma cells.Particularly preferred are mouse plasmacytoma cells that are deficientin the enzyme hypoxanthine-quanine phosphoribosyl transferase (HGPRT),which allows the selection of hybridomas from unfused antibody-producingcells or plasmacytoma cells when grown on a medium containinghypoxanthine, aminopterin, and thymidine.

It should be noted that the antibody-producing cell and the tumor cellcan be from different animal species. For example, see Nowinski et al.,Science, 210:534 (1980), which is incorporated herein by reference.

The hybridomas are then screened using acrosome-reacted human sperm orthe supernatant obtained from centrifuging acrosome-reacted human spermin known immunoassays to identify one or more hybridomas that producethe desired monoclonal antibody. Once the monoclonal antibody-producinghybridomas have been selected, the antibodies can be recovered from suchhybridomas by known techniques. Generally, it is useful to clone one ormore of the monoclonal antibody-producing hybridomas to expand it into acontinuous cell line that can be used to produce the monoclonalantibodies of the invention in quantity.

The previously mentioned method of producing the monoclonal antibodiesof the present invention is an in vitro method. The present inventionalso comprises an in vivo process for producing monoclonal antibodies tothe sperm antigen. Such antibodies are produced by placing a hybridomaof the invention intraperatoneally into a histocompatible orimmunosuppressed animal host, preferably a small mammal and mostpreferably a mouse. This causes the host to produce ascites tumorswhich, in turn, produce a fluid that contains monoclonal antibodiesproduced by the hybridoma. After sufficient time has passed for theantibodies to have been produced in sufficient quantities, they arerecovered by known techniques. This is particularly useful forfurnishing the monoclonal antibody in commercially useful quantities.

The present invention also includes hybridomas and continuous cell linesthat produce the monoclonal antibodies of the invention. Preferably, thehybridomas and cell lines produce monoclonal antibodies to anintra-acrosomal antigen that remains associated with the inner and outeracrosomal membranes after the acrosome reaction. Most preferably, thecontinuous cell lines have the characterstics of the hybridoma cell linehaving ATCC excession No. HB 10039 or mutants or variants thereof. Theinvention also encompasses individual cells within these cell lines.

These techniques can be applied to the production of a correspondingmonoclonal antibody to the non-human primate version of theintra-acrosomal antigen by the substitution of non-human primate spermfor the human sperm. Accordingly, such monoclonal antibodies as well astheir hybridomas and cell lines are within the scope of the invention.

A person skilled in the art can use known techniques to produce mutantsor variants of ATCC HB 10039. Such mutants or variants are encompassedwithin the present invention as long as they produce monoclonalantibodies reactive with the intra-acrosomal antigen of the invention.In addition, a person skilled in the art, using known techniques and theteachings disclosed herein, will be able to produce monoclonalantibodies reactive with the antigen of the invention but havingslightly different characteristics from ATCC HB 10039 or the antibodiesproduced by such cell line. Nevertheless, such monoclonal antibodies andthe hybridomas or cell lines that produce them are within the scope ofthe present invention. In a particularly preferred embodiment, themonoclonal antibodies produced by ATCC HB 10039 will prevent suchmonoclonal antibodies from reacting with the antigen of the invention.

The antigen of the invention, preferably SP-10, can be used to makemonoclonal antibodies reactive with epitopes different from the epitopeto which MHS-10 reacts. The purified or substantially purified antigencan be used as the immunogen for injecting into the host as previouslydescribed in the method for making the monoclonal antibodies of theinvention.

Since a variety of different systems and methods might be used toproduce a monoclonal antibody reactive with the human sperm antigen ofthe invention, a variety of monoclonal antibodies may result from thesemeasures that are distinct from the antibody illustrated in the examplesbelow. However such monoclonal antibodies, whose production is enabledby the teachings herein, still clearly within the scope of thisinvention. The salient feature of such antibodies, for the purposes ofthis invention, besides their monoclonality, is their reactivity in anyway with the human sperm antigen of the invention, regardless of thespecies of origin, isotype, molecular specificity, affinity, method ofproduction, or particular type of hybridoma employed in theirproduction.

The monoclonal antibody of the present invention may be purified by theuse of known techniques in view of the teachings contained herein. Forexample, ascites fluid containing the monoclonal antibody is mixed witha fractionating material, such as ammonium sulphate, to precipitateimmunoglobulins, including the monoclonal antibody of the invention. Theprecipitate is separated and resuspended in solution. The solution isdialyzed through a membrane to remove the fractionating material,producing a dialysate that contains the monoclonal antibody. Thedialysate is then run through an affinity column, such as a protein ASepharose bead column. The column is washed, and the antibody is elutedby lowering the pH by the use of an appropriate buffer.

The invention also comprises polyclonal antibodies to the humanintra-acrosomal sperm antigen. Such antibodies are produced by knowntechniques, appropriately modified in view of the teachings containedherein. An appropriate amount of the antigen is administered to ananimal host to create an immunogenic response. Any host that producesantibodies may be used. Conventionally used animals include rabbits androdents, such as rats or mice.

Once the animal has been immunized and sufficient time has passed for itto begin producing antibodies, polyclonal antibodies may be recovered bytechniques known in the art. The general method comprises removing bloodfrom the animal and separating the serum from the blood. The serum,which contains antibodies to the antigen, may be used as an antiserum.Alternatively, the antibodies can be recovered from the serum. Affinitypurification is a preferred technique for recovering purified orsubstantially purified polyclonal antibodies to the human sperm antigenof the invention.

The preferred method of producing the antigen of the invention is byculturing a proycaryotic cell, such as a bacterium, fungi, or othermicroorangism, or a eucaryotic cell, such as a yeast or a mammalian cellor cell line, that has been transformed by an expression vector or viruscontaining DNA that codes for the antigen or any desired part thereof.Preferably, the transformed cell is E. coli or a cell from a Chinesehamster ovary cell line. The expression vector contains a DNA sequence(molecule) that codes for the antigen or any desired part thereof whichhas been operably linked to the appropriate regulatory control nucleicacid sequences so that the DNA sequence can be expressed in thetransformed cell of choice.

The DNA of the invention is an isolated or substantially purified DNAsequence (i.e., polydeoxyribonucleotide) encoding a polypeptide thatcomprises the antigen of the invention. As used herein, the term"isolated" and variations thereof means that the DNA is in isolationfrom DNA encoding proteins naturally accompanying this antigen. Thus,the DNA of the invention includes DNA encoding the antigen when that DNAhas been cloned into a bacterial vector, such as a plasmid, or into aviral vector that may be harbored as a bacteriophage, provided that suchclones are isolated from clones that contain DNA encoding other proteinsnormally accompanying the antigen. As used herein, the term"substantially pure" and variants thereof means that the DNA issubstantially free of DNA and RNA that does not encode the antigen ofthe invention. That is, there will be no more than about 5 percent ofother DNA and RNA and preferably no more than about 1 percent of otherDNA and RNA in any sample that contains the DNA encoding the antigen ofthe invention. Preferably, the DNA of the invention is a complimentaryDNA (cDNA).

The cDNA of the invention is isolated from a testes cDNA expressionlibrary, using known techniques and the disclosure contained herein. SeeChang et al., Science, 240:324-326 (1988), incorporated herein byreference. An example of such a library is the lambda gt11testes-specific cDNA library available from Clonetech, Inc. Such alibrary can be screened with the monoclonal antibody of the invention,using known immunochemical techniques. This permits the identificationand subsequent isolation, purification, and sequencing of the cDNA. TheSP-10-5A cDNA was sequenced using a Sequenase sequencing kit (U.S.Biochemical Corp.) utilizing the Sanger dideoxy termination procedure(Sanger et al., Proc. Natl. Acad. Sci. 74:5463-5467 (1977), incorporatedherein by reference).

The genomic DNA of the invention is obtained through the application ofknown techniques in view of the teachings contained herein. Blotscontaining human genomic DNA digested with various restriction enzymeshave been probed separately with fragments containing the 5' and 3' endsof the SP-10-5 cDNA. The 5'SP-10-5 probe hybridized to a single band of4.5 kb while the 3' probe hybridized to a 7 kB band. This simple bandingpattern suggests that SP-10 is coded for a by a single copy gene or morethan one copy arranged as tandem repeats.

A human leukocyte genomic DNA library (Clontech) was screened with a 634bp fragment of SP-10-5. Five out of 5×10⁵ plaques showed stronghybridization to SP-10-5. These plaques were purified and shown to alsohybridize to the 3' end of SP-10-5, which suggests these clones containthe entire coding region of SP-10. Chromosomal location studies indicatethat the gene for SP-10 is located on chromosome 11, probably in thearea of the 11q2 band.

In a particularly preferred embodiment of the invention, the cDNA ishuman cDNA which codes for expression of the particularly preferredantigen of the invention, human SP-10, as shown in FIGS. 11A & B. HumanSP-10 is a 265 amino acid protein with the derived amino acid sequenceshown in FIGS. 11A & B. Preferably, it is coded for by the nucleotidesdesignated 61-855, although certain other nucleotides that code for thesame amino acids are known to those skilled in the art and could besubstituted for the nucleotides shown in the figures. The entire cDNA isalso shown and is approximately 1.35 Kb. The figures also show a 246amino acid SP-10 protein variant that results from alternative splicingof the mRNA. This protein is preferably coded for by the nucleotidesdesignated 61-556 and 614-855. The entire cDNA for this variant is alsoshown in FIGS. 11A & B and is approximately 1.29 Kb in length.

The invention also comprises immunogenic fragments of the human SP-10proteins. One of these is a 71 amino acid fragment extending from aminoacid 143 to amino acid 213 that is believed to contain the epitoperecognized by the MHS-10 monoclonal antibody produced by ATCC HB 10039.This peptide spans a domain of the protein that contains several repeatmotifs that have a hydrophilic character. It is coded for by a 214 basepair fragment shown as nucleotides 487-699 in FIGS. 11A & B. Thisepitope on the SP-10 variant protein comprises the amino acidsdesignated 143-165 and 185-213, preferably coded for by the nucleotides487-556 and 614-699.

Another preferred fragment of human SP-10 is the fragment containingboth the epitope and the carboxyl terminus region of the protein. Thispolypeptide preferably comprises the amino acids designated 143-265 inFIGS. 11A & B. It is most preferably encoded by nucleotides 487-855shown in FIGS. 11A & B. This fragment in the SP-10 variant is comprisedof the amino acids designated 143-165 and 185-265 in FIGS. 11A & B. Thisamino acid sequence is preferably coded for by the nucleotidesdesignated 487-556 and 614-855 in FIGS. 11A & B.

A third and most preferred fragment is the carboxyl terminus region ofthe SP-10 protein and its variant. Preferably, this fragment comprisesthe amino acids designated 177-265 in FIGS. 11A & B. Most preferably,this amino acid sequence is encoded by nucleotides 589-855. Thisfragment in the SP-10 variant is comprised of the amino acids designated185-265 in FIGS. 11A & B. The amino acid sequence is preferably encodedby nucleotides 614-855. In view of the fact that this region in monkeySP-10 was unexpectedly found to be 99% homologous with the human SP-10carboxyl region, this fragment is believed to contain functionallyessential epitopes and to be particularly useful as a contraceptivevaccine. Accordingly, the invention includes any immunogenic polypeptidethat is 99% homologous to the carboxyl terminus fragment of human SP-10.

In an alternative preferred embodiment of the invention, the cDNA isnon-human primate cDNA which codes on expression for non-human primateSP-10. Monkey, more particularly baboon and macaque, SP-10 cDNA andprotein are shown in FIGS. 16 and 19, respectively. Macaque SP-10 is a285 amino acid protein with the derived amino acid sequence shown inFIG. 19. It is 85% homologous to the human SP-10 shown in FIGS. 11A & B.Preferably, this protein is coded for by the nucleotide sequencedesignated 72-929 in FIG. 16. The entire cDNA sequence is approximately1.2 Kb in length. This sequence is 89% homologous to the human cDNAsequence shown in FIGS. 11A & B. The figures also show a monkey SP-10variant protein comprised of 251 amino acids. This protein is also theresult of alternative splicing of the mRNA as in the human protein.Preferably, it is coded for by nucleotides 72-582 and 685-929 as shownin FIG. 16. The entire cDNA for this variant is approximately 1.1 Kb inlength.

The invention also comprises immunogenic fragments of the monkey SP-10proteins. Preferably, the fragment comprises the carboxyl terminusregion of the protein. Most preferably, this fragment comprises eitherthe amino acid sequences designated 197-285 or 205-285 of FIG. 19 forthe monkey SP-10 protein and its variant. Most preferably, these aminoacid sequences are encoded by the nucleotides 660-929 or 686-929,respectively, as shown in FIG. 16. As previously mentioned, thisfragment is 99% homologous with the corresponding human carboxylterminal fragment. This high degree of homology provides strong evidencethat this is the primary functional region of the SP-10 protein and thatthis fragment of either the human or other primate SP-10 protein will bea particularly good candidate as an immunogen in a contraceptive vaccinefor humans or other primates.

It will be recognized by persons skilled in the art that the cDNAsequence of the preferred antigen may be modified by known techniques inview of the teachings disclosed herein. For example, different codonscan be substituted that code for the same amino acid as the originalcodon. Alternatively, the substitute codons may code for a differentamino acid that will not affect the antifertility activity orimmunogenicity of the antigen or which may improve the antifertilityactivity or immunogenicity of the antigen. For example, site directedmutagensis or other techniques to create single or multiple mutations,such as replacements, insertions, deletions, and transpositions, asdescribed in Botstein and Shortle, "Strategies and Applications of InVitro Mutagenesis," Science, 229:193-1210 (1985), which is incorporatedherein by reference, can be employed. Since such modified DNA can beobtained by the application of known techniques to the teachingscontained herein, such DNA is within the scope of the claimed invention.

Moreover, it will be recognized by those skilled in the art that a cDNAsequence obtained from a cDNA expression library or prepared fromisolated messenger RNA that codes for the antigen of the invention mayexhibit the natural allelic variations found among individuals. Sincesuch variant cDNA sequences are obtained by the teachings containedherein, they are within the scope of the invention.

Finally, it will be recognized by those skilled in the art that the cDNAsequence (or fragments thereof) of the invention can be used to obtainother cDNA sequences that hybridize with it under conditions of highstringency, using general techniques known in the art, or used to obtainany DNA that hybridizes with the cDNA under conditions of highstringency. Such DNA includes any genomic DNA. Accordingly, the DNA ofthe invention includes DNA that shows at least 75 percent, preferably 90percent, and most preferably 95 percent homology with the genomic DNAcoding for the antigen SP-10 or the cDNA of FIGS. 11A & B and 16,provided that such homologous DNA encodes the antigen of the invention.

The DNA of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform a microorganism for the expression and production of theantigen of the invention. Such techniques include those disclosed inU.S. Pat. Nos. 4,440,859 issued Apr. 3, 1984 to Rutter et al., 4,530,901issued Jul. 23, 1985 to Weissman, 4,582,800 issued Apr. 15, 1986 toCrowl, 4,677,063 issued Jun. 30, 1987 to Mark et al., 4,678,751 issuedJul. 7, 1987 to Goeddel, 4,704,362 issued Nov. 3, 1987 to Itakura etal., 4,710,463 issued Dec. 1, 1987 to Murray, 4,757,006 issued Jul. 12,1988 to Toole, Jr., et al., 4,766,075 issued Aug. 23, 1988 to Goeddel,et al., and 4,810,648 issued Mar. 7, 1989 to Stalker, all of which areincorporated herein by reference.

The DNA of the invention may be joined to a wide variety of other DNAsequences for introduction into an appropriate host. The companion DNAwould depend upon the nature of the host, the manner of the introductionof the DNA into the host, and whether episomal maintenance orintegration is desired.

Generally, the DNA (preferably the cDNA) is inserted into an expressionvector, such as a plasmid, in proper orientation and correct readingframe for expression. If necessary, the DNA may be linked to theappropriate transcriptional and translational regulatory controlnucleotide sequences recognized by the desired host, although suchcontrols are generally available in the expression vector. The vector isthen introduced into the host through standard techniques. Generally,not all of the hosts will be transformed by the vector. Therefore, itwill be necessary to select for transformed host cells. One selectiontechnique involves incorporating into the expression vector a DNAsequence, with any necessary control elements, that codes for aselectable trait in the transformed cell, such as antibiotic resistance.Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

The preferred expression vectors for use in procaryotic cells are pWR590 (Guo et al., Gene 29:251-254 (1984), incorporated herein byreference), pGEX2T (Pharmacia), and pMAL-C (Maina et al., Gene74:365-373 (1988), incorporated herein by reference). Preferably, a tacpromotor under the control of a lac repressor is used. The promotorincludes a ribosome binding site, and the repressor is coded for by thelac I^(Q) gene.

For eucaryotic cells, there are two preferred expression vectors. One isthe baculovirus vector pAc373 under the polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus in Spodopterafrugipenda cell lines. The other is the yeast expression vector pYEUra3under the Ga11 and Ga110 promoters.

The antigen of the invention or an immunogenic fragment thereof may beused as an immunogen in an antifertility vaccine for humans and otherprimates and other mammals. Such a vaccine can be prepared by techniquesknown to those skilled in the art and would comprise, for example, theantigen, a pharmaceutically acceptable carrier, an appropriate adjuvant,and other materials traditionally found in vaccines. An immunologicallyeffective amount of the antigen or fragment thereof is determined bymeans known in the art. In view of the close homology between the monkeyand human SP-10, it may be desirable to use the monkey SP-10 in humansif it were more immunogenic than human SP-10. To the extent that animmunogenic fragment is used, it is expected to include the carboxylterminus region of SP-10.

The cost effectiveness of expression and scale up of eukaryotic proteinsin E. coli has made this the model of choice for initial expression ofthe SP-10 recombinant vaccine. Many low molecular weight proteinsexpressed in E. coli may be rapidly degraded unless these proteins arefused to a large E. coli protein. (We have used the pWR590 expressionsystem (Guo et al, Gene 29 (1984), 251-254 incorporated herein byreference) which includes the E. coli lac promotor and a portion of thecoding sequence for beta-galactosidase which can code for approximately590 amino acides. The injection of the SP-10 protein attached to thisattenuated beta-galactosidase has proven to be strongly immunogenic inrabbits.) In the preferred embodiment genes encoding portions of theentire open reading frame for the SP-10 protein are inserted into abacterial plasmid containing a strong promotor and a bacterial gene orportion thereof. The expression of the SP-10 protein occurs inconjunction with the bacterial protein and the two proteins are attachedthrough amino acid linkages. Following lysis of the bacteria andpurification of the resulting fusion protein using preparative SDS-PAGEor other methods, such a "fusion protein" may afford the advantage thatthe bacterial protein may function as a adjuvant to enhance the immuneresponse of the host to the recombinant eucaryotic antigen.

The antigen of the invention may also be used in quantitative assayswith the monoclonal antibody of the invention for the detection ofacrosome-reacted sperm. The purified antigen may be used as a standardor a control. Direct or indirect immunofluorescence can be used tocorrelate the extent to which human sperm in a sample have undergone theacrosome reaction. This data can be used to develop a standard curvewhereby the extent to which human sperm in a sample have undergone theacrosome reaction can be determined by competition or direct immunoassayof SP-10 or other intra-acrosomal antigen obtained from the culturesupernatant of the acrosome-reacted sperm and assayed byradioimmunoassay or enzyme-linked immunoassay.

The antigen of the invention can be used to detect and measure antispermantibodies. The detection of antisperm antibodies has witnessed an arrayof assay methods over the years, including assays based on agglutinationof sperm cells (Kibrick assay), cytotoxicity and immobilization of spermcells, (Isojima), binding to sperm surface or cytoplasmic antigens (Herret al, 1987, Am. J. Reprod. Immunol. 11:75-81) using ELISA or RIAformats or the binding of class specific immunobeads (Bronson). Theseassays are distinguished by the fact that they are targeted to wholecells. To date there has not been a widely used assay for antispermantibodies that employs single molecular target antigens or groups ofthese targets antigens. The advent of recombinant methodologies opensthe possibility that defined sperm antigens might now be employed astargets for measuring antisperm antibodies. SP-10 is an example of anantigen that may work in this regard.

The antigen of the invention, including polypeptides encoded by the cDNAof FIGS. 11A & B and 16, fragments, modifications and derivativesthereof, is also useful as reagents in studying fertility andcontraception in mammals, especially primates, to better understandhuman fertility and infertility. The carboxyl terminal region of humanor monkey SP-10 is expected to be particularly useful as a reagent forthe further study of fertility, infertility, and contraception. Becauseof the high degree of interspecies homology in this region, it isthought that this is a functional domain of the protein. Betterunderstanding that functional domain will lead to a better understandingof the SP-10 protein itself and the role it plays in primate sperm. Theuse of such polypeptides as a laboratory reagent is readily within theskill of those skilled in the art who engage in such activities. Thecarboxy terminal region will also be useful for making monoclonalantibodies that can also be used as laboratory reagents in the furtherstudy of SP-10 and human fertility.

The monoclonal or polyclonal antibodies of the present invention may beused to identify sperm, particularly acrosome-reacted sperm, in varioustypes of samples, such as human semen. The antibodies may be used as areagent in known immunoassays for determining the presence orconcentration of human sperm, sperm heads, or acrosome-reacted sperm.Such immunoassays include, but are not limited to, radioimmunoassay,competitive ELISA, immunoprecipitation assay, enzyme-linkedimmunoabsorbent assay, and direct or indirect immunofluorescence assay.One application of this approach is the identification of sperm insexual assault evidence.

A composition for determining the presence or concentration of humansperm or otherwise evaluating sperm in accordance with the presentinvention contains a concentration of the antibody effective to detectthe presence of such material or quantify its amount. The antibody canbe mixed with or attached to any suitable carrier, such as a latexparticle, a plastic bead, or a plastic microtiter plate. It may also beconjugated with an enzyme or dye or radiolabeled, depending upon whatimmunological method is employed.

The monoclonal or polyclonal antibodies of this invention are alsouseful for the isolation or purification of human sperm from complexmixtures or solutions on the basis of a selective immunologicalreaction. The mixture is brought into contact with immobilizedantibodies of the invention, which will separate the sperm from themixture by forming immobilized complexes of the sperm bound to theantibody. When the mixture is removed, the sperm is separated from theantibodies and recovered in purified form by known techniques.Preferably, the sperm is permeabilized before being contacted with theantibodies.

A composition in accordance with the invention useful for purifying orremoving sperm from complex mixture contains an effective amount of themonoclonal or polyclonal antibody of this invention, immobilized on anacceptable matrix or admixture with an acceptable carrier, to permitreaction and binding with the sperm.

The antibodies of this invention are also useful reagents for researchinto the structure, function, and immunochemistry of human sperm,particularly acrosome-reacted human sperm. A composition in accordancewith the present invention useful as an investigational reagent containsan amount of antibody effective to provide the information upon mixturewith the sample and subsequent analysis. Determination of the amount ofantibody necessary to accomplish a particular research goal depends uponthe specific types of investigation involved and is readily within theskill of one engaged in such research.

The monoclonal or polyclonal antibody of the invention, is preferablyused in the following diagnostic procedures: (1) as a probe for immaturegerm cells in semen in order to detect infertility caused by defects inhuman spermatogenesis; (2) as a marker for the acrosome reaction inhuman sperm; (3) as the active ligand in a sperm cell affinity bead forisolation of sperm cells for (a) purification of sperm cell DNA forsubsequent RFLP analysis with application in forensic science andpaternity testing and (b) isolation of acrosome-reacted human sperm forsubsequent fertilization of human eggs; and (4) as a probe foridentifying sperm heads in material evidence obtained from sexualassaults.

Round cell syndrome refers to the presence of many round cells in semenin addition to spermatozoa. These round cells may be lymphocytes,macrophages, sloughed epithelial cells from accessory sex organs, andgerm cells which have not fully matured into spermatozoa. Currently,there are no immunohistochemical probes which selectively identifyimmature germ cells during analysis of round cells present in semen.

The present invention will allow the numbers of immature germ cellspresent in a semen sample to be determined. This will permit accuratedetection of cases of premature or excessive sloughing of germ cells andhence identify cases where problems are occurring with the process ofspermatogenesis. This may be of significance in cases wherespermatogenesis is being interfered with by environmental toxins,infections of the male reproductive tract, or alterations in the normalhormonal balance of the male. Thus, the invention will allow for adifferential diagnosis of round cells in semen, giving a positiveidentification of some early stages of germ cells.

This will be accomplished by contacting a sample containing human spermwith the monoclonal or polyclonal antibody of the invention, where theantibody has been labeled by known techniques with a detectable entity.The image formed by the antibodies is then evaluated and compared toknown or standard images of human sperm at the appropriate state ofdevelopment.

The monoclonal or polyclonal antibodies of the invention will also beuseful as a marker for the acrosome reaction in human sperm. They couldbe used to assess the number of acrosome reactive or unreactive sperm ina given population. The antibody is contacted with a sample of humansperm for a time and under conditions sufficient for the antibody in anyacrosome-reacted sperm to form an antigen-antibody complex. The complexis then detected by known techniques for detecting the label ordetectable moiety attached to the antibody. These include direct orindirect immunofluorescence, radioimmunoassay, or enzyme-linkedimmunoassay. This application may be of use to diagnose infertility,when such infertility is due to defects in the sperm's ability toacrosome react as well as defects in the rate of acrosome reaction.

Currently, forensic laboratories and labs concerned with paternitytesting are relying on the powerful techniques of RFLP analysis toidentify potential suspects or identify the correct father of a givenchild. Utilizing DNA obtained from material evidence from victims, fromthe crime scene, or from the blood of possible parents, the DNA is cutwith restriction enzymes and electrophoresed. DNA probes which recognizea series of specific nucleotide sequences within the human genome arethen employed to identify specific genetic polymorphism, thusidentifying DNA from the crime scene, victim, or suspect or from aparent and a child. One current problem in this field of DNAfingerprinting of sexual assault evidence is the isolation of sperm DNAfrom the other cellular materials obtained from a sexual assault victim.Cells (such as bacteria, yeast, or cervical, anal, or oral epithelialcells) may contaminate the specimens. The monoclonal or polyclonalantibodies of the invention conjugated to a bead may be used to enrichfor sperm cells in such mixtures and thus allow for selective extractionof human sperm DNA.

The acrosome reaction is a necessary prerequisite to fertilization. Itis thought that only acrosome reacted sperm can fertilize eggs. Sincethe antigen of the invention appears to be displayed on the inneracrosomal membrane of acrosome-reacted sperm, an antibody-bead conjugatemay be used to selectively adsorb acrosome reacted sperm onto a bead orsuitable cell affinity matrix. The sperm might then be removed from thebead or used on the bead, to interact with and fertilize human eggs.

Thus, the invention provides a means for isolating acrosome-reactedhuman sperm cells. Immobilized monoclonal or polyclonal antibodies ofthe invention are contacted with a sample containing human sperm cellsfor a time and under conditions sufficient for the antibody to bind tothe sperm cells to form antibody-sperm cell complexes. The complexes arethen removed from the sample. Preferably, the bound complexes arewashed. The sperm cells are then separated from the antibodies usingknown techniques to provide the sperm cells as an isolate. Preferably,the antibody is attached to an immunoaffinity bead.

Such a bead might be used as a vehicle to administer a selectedpopulation of acrosome-reacted sperm into the uterus or oviduct ofinfertile women who otherwise ovulate normally but are diagnosed ashaving "unexplained infertility" (possibly of an immune origin). Thiswould allow for a laboratory technician to circumvent in vivocapacitation and present the woman with a population of acrosome-reactedsperm. Further, the acrosome reacted sperm isolated from the affinitybead might be used in in vitro fertilization with human eggs.

Often sexual assault evidence contains few sperm cells. This is oftendue to the fact that the evidence is the eluate from a dried swab of abody cavity, resulting in sperm heads which have detached from theirtails. The specificity of the monoclonal antibody of the invention forsperm heads and its lack of cross reactivity with other human cell typesallows the probe to be employed in analysis of sexual assault evidenceto prove the existence of sperm cells.

The monoclonal antibody of the invention is also expected to be usefulas an active ingredient in a contraceptive gel, cream, or othercomposition. An amount effective to create an antifertility effect in ahuman or other mammal is mixed or otherwise added to a pharmaceuticallyacceptable carrier, which can then be administered for contraceptivepurposes.

The DNA of the invention is useful not only in the preparation of theSP-10 protein but as probes for finding the DNA of other mammalianspecies that codes for SP-10 in those species. Thus, it is a useful toolfor the preparation of contraceptive vaccines for other species.Moreover, the cDNA of the invention is useful as a laboratory reagent,in particular a probe for finding the SP-10 gene in primate or othermammalian genomic libraries or for mapping the SP-10 gene to itschromosome.

Although the various utilities for the antigens, polypeptides,monoclonal antibodies, and DNA molecules of the invention may have beendescribed above with reference to humans, they also apply to otherprimates in view of the experimental results regarding macaque andbaboon SP-10 protein and cDNA and the other teachings disclosed herein.

It is to be understood that application of the teachings of the presentinvention to a specific problem or environment will be within thecapabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention, processes for their production, and processes for the useappear in the following examples.

EXAMPLE 1 Preparation of Monoclonal Antibody MHS-10

Monoclonal antibodies were produced using the procedure of Galfre, etal., Nature, 266:550 (1977), incorporated herein by reference. Balb/cfemale mice were immunized four times with 1×10⁷ thrice washed humansperm in incomplete Freunds adjuvant. Each immunization amounted tothree injections of 0.1 ml each, injected intramuscularly andintraperitoneally. The sperm were obtained from blood type O donors.After fusion of the mouse spleen cells with myeloma cell line SP2/0(Schulman, et al., Nature 279:269 (1978), incorporated herein byreference), cells were distributed into 96 well plates containing HATselection medium. HAT selection medium comprises hypoxanthine,aminopterin, and thymidine. Enzyme-linked immunosorbent assay (ELISA)screening for antibodies was performed 14 to 21 days after fusion byemploying 1×10⁵ sperm target cells per well. Hybridomas that elicitedpositive binding to sperm were expanded and cloned by the limitingdilution method of Galfre et al. Twenty-five stable IgG secretingantisperm hybridoma lines were established.

The antibodies were then tested by indirect immunofluorescence for theirability to bind to human sperm. Indirect immunofluroescent localizationof the MHS-10 antigen on ejaculated human spermatozoa was performedaccording to the methods of Herr et al., Biol. Reprod., 32: 695-711(1985), incorporated herein by reference. One of these hybridomas, ATCCHB 10039, produced an antibody of the immunoglobulin in subclass IgG1that bound to the acrosome of fixed, permeabilized human sperm.

EXAMPLE 2 Hamster Egg Penetration Test

The MHS-10 antibody has been shown to block the interaction of humansperm with zona free hamster eggs. See Anderson et al., J. Reprod.Immunol. 10:231 (1987) and Yangimachi et al., Biol. Reproduction 15:471(1976), incorporated herein by reference. In this test, the zonapellucida of hamster eggs was dissolved by treatment with a protease,and human sperm were subsequently added to the zona free eggs. Theability of the sperm to bind and enter the egg was scored by countingthe sperm nuclei lying within the egg cytoplasm. Using this technique,the MHS-10 antibody was found to inhibit the number of sperm interactingwith hamster eggs.

EXAMPLE 3 Purification of MHS-10

The antibody was first precipitated as follows. Add 8 mls ice coldsaturated ammonium sulfate slowly, dropwise with stirring, to 10 mlsascites fluid representing 380-400 mg total protein. This is allowed tostir in the cold for 3 hours. Centrifuge at 10,000 rpm in Sorvall RC-5Bfor 20 minutes. Discard supernatant and resuspend pellet inapproximately 5 ml dH20. Dialyse for 48 hours at 4° C. against 4 changesof PBS at pH 8.0

A protein A column was prepared as follows. Swell 3 gm protein Asepharose CL-4B (Pharmacia) in about 50 mls PBS pH 8.0 for 20 minutes.Pour swollen gel into disposable syringe fitted with a teflon supportand 3-way stopcock. Allow all the excess buffer to be excluded as thecolumn packs by gravity. When all the gel is in the column, wash with atleast 50 ml buffer (PBS pH 8.0). Store in PBS and 0.2% sodium azide at4° C. until ready for use.

The antibody was purified on the protein A column as follows. Mix 2 mldialysed saturated ammonium sulfate precipitate (representing 4 mlsascites) with protein A Sepharose beads in PBS pH 8.0 and agitate endover end overnight at 4° C. Pour the beads into the column and washthrough unbound material with PBS pH 8.0 until the baseline on the UVmonitor is flat. Bump with PBS pH 5.5 to elute bound IgG1 antibody. Pumpbuffer through column at a low flow rate, 0.6 ml/min, since antibodyelutes slowly at this pH. Clear column of remaining bound material,including other isotypes of antibody with 0.01M Citrate buffer pH 3.0with 0.87% NaCl. Finally, re-equilibrate column with PBS pH 8.0 andstore with 0.2% sodium azide at 40° C.

A typical purification started with 10 mls ascites fluid representing380-400 mg total protein. After the SAS precipitate is resuspended in 5mls dH₂ O and dialyzed, the total volume will be approximately 7 mls andthe protein concentration 15 mg/ml.

From 75 mg dialyzed SAS precipitate purified on a protein A column, 43mg will be proteins other than IgG that do not bind to the column(Fraction I), and 32 mg will be pure IgG that elutes with the pH 5.5 PBS(Fraction II). A small amount of immunoglobulin of different subclassesis cleared from the column with the pH 3.0 citrate (Fraction III).

Reloading Fraction I onto the column and eluting again did not yield anyadditional IgG, indicating that the binding capacity of the column wasnot exceeded initially with the 75 mg SAS precipitate.

A 10% acrylamide gel was run to confirm the identity of the purifiedfraction. When 100 ug of protein from the starting ascites, Fraction I,and Fraction II were run on the gel and stained with Comassie Blue, onlytwo bands of heavy and light chain antibody were present in Fraction IIand little or none was apparent in the Fraction I of material that didnot bind to the protein A column.

EXAMPLE 4 Preparation of Monoclonal Antibodies

Monoclonal antibodies reactive with the head of acrosome reacted spermmay be obtained by immunizing mice with the supernatant resulting fromseparation of acrosome-reacted sperm from the products of the acrosomereaction. A number of methods may be employed to acrosome react humansperm, including incubation with ionophores, follicular and oviductalfluids, and soluble or intact zona pellucida. The sperm are centrifugedat low speed (50×g) to separate the sperm cells from soluble hybridvesicles consisting of outer acrosomal membrane and plasma membrane.This supernatant is employed in standard immunization protocols asoutlined in Example 1.

EXAMPLE 5 Purification of SP-10

An affinity column was prepared as follows. Cyanogen bromide activatedsepharose 4B (Sigma Chemical Co.) was used as the immobilizing phase forthe MHS-10 antibody. To prepare the beads, 3.0 g of dry beads wereswollen in 1 mM HCl for 15 minutes and then washed in 200 ml of thesame. Swollen volume was about 10 ml. The beads were washed withcoupling buffer (0.1M NaHCO₃, pH 8.3 with 0.5M NaCl) and immediatelytransferred to 15 ml solution of 32 mg purified MHS-10 in couplingbuffer. The mixture was agitated end over end in a 50 ml tube overnightat 4° C.

The beads were then washed free of any unbound material with 100 mlcoupling buffer. Unreacted active sites on the beads were blocked byincubating with 0.1M tris, 0.1M glycine pH 8.3 with 0.5M NaCl for 2-3hours at room temperature.

The column was prepared in a 12 ml syringe with teflon support and 3-waystopcock and washed with coupling buffer again. It was then washedalternately with 0.1M acetate, 0.5M NaCl pH 4.0, followed by couplingbuffer, then acetate buffer, and finally equilibrated with 0.1M Hepes pH8.0 with 0.2% azide for storage.

A BCA protein assay on the material that did not bind to the beadsindicated that 2 mg of the original 32 mg did not bind.

The sperm was prepared as follows. Fresh ejaculates were allowed toliquefy for 1 hour and then washed twice with 40 ml Ham's F10 mediumwith Hepes buffer pH 8.0 by centrifuging at 600×g and discarding thesupernatant. Sperm pellets were stored frozen at -80° C. with proteaseinhibitors (5 mM benzamidine, 1 mM PMSF, 2 ug/ml leupeptin, 2 ug/mlpepstatin).

Prior to purification of antigen, sperm pellets were thawed and douncehomogenized in minimum volume of the buffer in which they were frozen.

The extract of soluble proteins was centrifuged at 10,000×g in themicrofuge. Preliminary results indicated that the yield of antigen maybe further increased by re-extracting the pellets in 1% SDS and poolingwith the initial soluble extract such that the final SDS concentrationis 0.25%.

The antigen was purified with an affinity column as follows. Spermextract is either passed over a 10 ml column with sepharose 4B beads oragitated with the beads overnight at 4° C. to preabsorb any proteinswhich would nonspecifically bind to the beads themselves. The extractwas loaded onto the top of the affinity column, and allowed torecirculate over the column by pumping at 0.6 ml/min overnight at 4° C.Unbound material (Fraction I) was washed from the column with 0.1M Hepesbuffer pH 8.0 until baseline on UV monitor was flat. Enriched antigen(Fraction II) was bumped from column with 0.1M Glycine buffer pH 2.2with 0.87% NaCl, and fractions were collected until the baseline wasagain flat. Finally, the column was reequilibrated with Hepes buffer pH8 and stored at 4° C. with 0.2% sodium azide. The enriched Fraction IImay be passed over a third column, which is the affinity column preparedwith goat anti-mouse IgG, to remove any MHS-10 antibody that may havebeen released from the MHS-10 affinity column.

The goat anti-mouse IgG affinity column was prepared with CNBr activatedsepharose in exactly the same way as the MHS-10 affinity columndescribed above with the following changes. To prepare a 5 ml column,1.5 g of dry beads were used. It was incubated overnight at 4° C. with1.5 mg goat anti-mouse IgG with minimum cross reactivity to human,horse, and bovine serum proteins (Jackson Immunochemical Laboratories,Inc.)

In a typical antigen purification, 40 sperm samples were thawed andextracted as described above. The total volume was 7.8 ml and 100 ul wasset aside to determine protein concentration and for gelelectrophoresis. Total protein was determined by the BCA procedure to be68 mg. The extract was preabsorbed with sepharose 4B-200 beads and theresulting volume of 38 mls was then allowed to bind to the affinitycolumn.

Proteins which did not bind to the affinity column were eluted with 0.1MHepes pH 8.0 and collected as Fraction I. After dialysis andlyophilization, this protein was redissolved in 1.0 ml PBS and the totalprotein was determined to be 16 mg.

Proteins which did bind to the affinity column were eluted with 0.1Mglycine buffered saline pH 2.2 and collected as Fraction II. The totalvolume of Fraction II was 20 mls. Half of this fraction was furtherabsorbed against the goat anti-mouse IgG column to remove any mouseantibody that might have been released from the affinity column. Afterdialysis and lyophilization, each half of this purified fraction,absorbed and nonabsorbed, was redissolved in 100 ul PBS. The absorbedhalf of the fraction contained 39 ug of protein and the nonabsorbed halfcontained 64 ug of protein. Another 65 ug of protein was present in theFraction III eluted when the column was reequilibrated with Hepes bufferpH 8.

Other protein not accounted for could have been lost to the system andany of the various steps, including non-specific absorption to theprecolumn. The amount unaccounted for in this experiment seemedunusually high perhaps due to the increased number of manipulations. Inother experiments without the precolumn or anti-mouse IgG column, yieldsof approximately 150 ug of enriched Fraction II antigen from 12ejaculates would be typical.

To visualize the degree of enrichment of the antigen, a 10% minigel wasrun with 20 ug/lane of starting material sperm extract and the 3fractions collected from the affinity column. Upon Western blotting, noantigen was apparent in Fraction I, the material which did not bind tothe column. Both Fraction II and Fraction III revealed the full array ofantigen bands. Anomalous bands, apparently the result of mouse IgG beingreleased from the column itself, were apparent on the null ascitescontrol blot as well as in Fraction III and the non-absorbed FractionII. The anti-mouse IgG column removed most of this contamination in theabsorbed fraction.

Amido black staining of the blot or silver staining of the gel ofenriched Fraction II protein typically revealed two bands staining inthe MHS-10 region around 30 kD, two other bands around 50 kD and 66 kD,and a very heavy band around 78 kD. The bands higher than 30 kD wereconsidered to be contaminants because they were nonimmunoreactive.

The purified SP-10 antigen analyzed on 2-D gels displayed peptides withmolecular weights from 18-34 Kda. Peptide bands of 34, 26, 24, and 18 Kdare then purified to homogenity (90%-98%) by sequential electrophoresisand electroelution. A 10% SDS PAGE gel containing the SP-10 fractionfrom the affinity column is electrophoresed in one dimension and peptidebands corresponding to the immunoreactive antigen are identified byimmunoblot. SP-10 peptides are then cut as strips from the gel. The cutstrips are electrophoresed on 12% gels. The peptides are scanned forpurity. The peptides are transferred to nitrocellulose membranes byelectroelution and may then be elututed for inoculation or subsequentbiochemical analysis.

EXAMPLE 6 Purification of Antigen by Reverse Phase HPLC

Purification of SP-10 from Human Serum

Serum from 8 to 12 ejaculates was washed by centrifugation in 25 ml eachof Ham's F10 medium, Hepes buffer pH 7.4, two times at 550×g for 10minutes, and then stored frozen at -20° C. until needed. The sperm wasthawed, resuspended in 1-2 ml 0.1% TFA (trifluoroacetic acid), douncehomogenized to extract soluble antigen, and microfuged two times at13,000 ×g, then filtered through a 0.22 um filter to remove insolublematerial. The soluble extract containing 5-10 mg total protein wasfractionated on a Gilson HPLC with a Brownlee reverse phase column, 10mm×25 cm, packed with Aquapore C-8, 300 A pore size, 7 um silica bead.With a flow rate of 1.5 ml/min, a gradient of 0-80% Solvent B over 50minutes was run. Solvent A was 0.1% TFA in distilled water and solvent 3was 0.1% TFA in 2- propanol. Fractions corresponding to individual peakswere detected at 230 nm and collected manually.

Preparative gel electrophoresis

These fractions were lyophilized with a Savant Speed Vac, dissolved inLaemmli sample buffer and separated on a 10% polyacrylamide gel.Proteins were electroblotted for 40 minutes at 500 mAmps (10 mM CAPSbuffer, 10% methanol, pH 11.0) onto a PVDF membrane backed up with asecond PVDF membrane and a third nitrocellulose membrane to captureproteins passing through the PVDF. The PVDF membranes were stained withCoomassie Blue to identify the proteins present in each fraction whilethe nitrocellulose were probed with MHS-10 antibody to identify theantigenic bands to be cut from the PVDF blots for sequencing.

Amino acid sequencing

Amino acid sequencing was performed in the University of VirginiaProtein and Nucleic Acid Sequencing Facility. The N-terminal amino acidsequence was determined using an Applied Biosystems 470 A Gas PhaseProtein Sequencer. Dried samples of the MHS-10 immunogen were taken upin 75% formic acid and applied to a glass fiber filter coated withPolybrene. The filter was dried and applied to the sequencer. One cycleof Edman degradation was performed without phenylisothiocyanate (PITC)followed by twenty to thirty cycles with PITC. Cleavage of theN-terminal amino acids was accomplished via gas phase trifluoroaceticanhydride resulting in the formation of anilinothiazolinone derivatives.The PTH derivatives or a mixture of PTH standards was analyzed on Waters840 HPLC system with an IBM C18 reverse phase column and will bedetected at wavelengths of 254 and 313 nm. Two SP-10 peptides, the 3 kDand 18 kD forms, have been isolated and amino acid microsequenced.

The N-terminus sequence of the first 12 amino acids of the 30 kD bandwas found to be: XTVAEXTSGEXA. This sequence aligned with the predictedsequence deduced from cDNAs beginning with amino acid number 78. TheN-terminus sequence of the first 7 amino acids of the 18 kD band wasfound to be: XDEQXSG. This sequence aligned with the predicted sequencededuced from cDNAs beginning with amino acid number 140.

EXAMPLE 7 Characterization of the Antigen SP-10

Biochemical and morphological characterization of SP-10 shows an acidic,polymorphic protein which is conserved in the human population. Arisingduring spermatogenesis within the nascent acrosomes of developingspermatids and localizing within the acrosome of intact sperm, SP-10 isnot located on the plasmalemma but becomes exposed on the sperm surfacefollowing the acrosome reaction. SP-10 is thus a differentiation markerof acrosome development in the human and an example of anintra-acrosomal immunogen exposed prior to fertilization, offering apotential target for immunocontraception. SP-10 has been designated a"primary vaccine candidate" by the World Health Organization Taskforceon Contraceptive vaccines, due to its tissue specificity and evidencethat the MHS-10 monoclonal antibody inhibited the sperm/egg interactionin the hamster egg penetration assay.

Materials and Methods

1. Immunocytochemistry of Human Testis

Testes were obtained from elective orchiectomies for prostate carcinomafrom patients untreated with steroids. Testes were fixed in 2%formaldehyde in 0.1M phosphate buffer and embedded in paraffin. Tenmicron sections were mounted on gelatin coated microscope slides,deparaffinized in a graded series of ethanols and rehydrated inphosphate buffered saline (PBS). Sections were pretreated with 10%normal goat serum for thirty minutes, washed 3×in PBS, and reacted witha 1:1000 dilution of monoclonal antibody MHS-10 or control IgG₁ in 1%normal goat serum for 30 minutes. Following washing, sections weretreated with 1:100 dilution of goat anti-mouse IgG (JacksonImmunoresearch Laboratories, West Grove, Pa.) for 30 min, washed thriceand incubated with mouse peroxidase-anti-peroxidase, 1:200, in PBS for30 min, followed by thrice washing in PBS. Brown reaction product,indicating the location of the SP-10 antigen, was developed with 0.05%diaminobenzidine with 0.015% hydrogen peroxide.

2. Immunofluorescence Microscopy

Motile Sperm. Live ejaculated sperm were incubated 1.5 hr in RPMI 1640medium with 3.5% BSA at 37° with 5% CO₂. 1.5×10⁸ sperm were incubatedfor 1 hr with MHS-10 antibody at 1:100 or control IgG₁ at 1:100 dilutedin RPMI. Samples were washed 2×in medium and reacted with a 1:100dilution of goat anti-mouse IgG-FITC (Jackson Immuno ResearchLaboratories) for 1 hr. Samples were washed 2×an observed as wet mounts.Fifty percent of sperm were motile at time of addition of primaryantibody; 25% at addition of second antibody and approximately 10% weremotile at time of scoring 1000 motile cells.

Effect of Tx-100 or methanol permeabilization. 3×10⁸ sperm were washed3×in phosphate buffered saline (PBS) containing 2 uMphenylmethylsulfonylfluoride. Sperm were fixed 30 min in 3%paraformaldehyde. Aliquots were permeabilized with 0.5% Triton X-100 or100% methanol for 30 min at room temperature. Unpermeabilized sampleswere treated with PBS. After washing 2×, samples were incubated with a1:100 dilution of MHS-10 in PBS for 1 hr at 37° C., followed by a 1:100dilution of goat anti-mouse IgG. Preparations were washed 2×and mountedin 90% glycerol, 0.25M Tris, pH 7.5 and examined.

Routine method for scoring MHS-10 staining and acrosome reacted sperm.Based upon evidence (see results) that membrane permeablization exposedthe SP-10 antigen, the following standard method was developed. Spermfrom liquefied semen samples were washed twice in Ham's F10 mediumbuffered with 0.1M Hepes. For induction of the acrosome reaction, spermsuspensions were capacitated for 3 hrs at 37° in Biggers, Whitten &Whittingham (BWW) medium (Biggers et al., Methods in MammalianEmbryology (ed. J. C. Daniel) 1st ed., p. 86, (Freeman, San Francisco,1971), incorporated herein by reference) with 3.5% human serum albumin(HSA). Samples were acrosome reacted for 1/2 hr in 10 uM calciumionophore A23187 in BWW containing 0.3% HSA. Sperm were cytocentrifugedonto a microscope slide, allowed to air dry, and fixed with severaldrops of 3% paraformaldehyde for 45 min at room temperature. Slides weretreated with 100% methanol for 20 min at room temperature and blockedwith 10% normal goat serum (NGS) for 15 min. Slides were incubated witha 1:100 dilution of monoclonal antibody MHS-10 in 0.01M phosphatebuffered saline, pH 7.4, 1% NGS for 45 min at room temperature, followedby three washes in PBS. A 1/100 dilution of fluorescein isothyocyanateconjugated goat anti-mouse IgG (Jackson Immuno Research) in PBS wasemployed as a second antibody. Specimens were washed extensively and wetmounted in 90% glycerol, 10% 0.1M Tris, pH 7.5 with orthophenylenediamine added to prevent fading of fluorescence.

3. EM Immunocytochemistry

Testis tissue was fixed in 2% glutaraldehyde, 2% formaldehyde in 0.Mcacodylate buffer, pH 7.3. A portion was post-fixed in 2% osmiumtetroxide. Tissue was embedded in Araldite 502. Gold sections were cuton an ultramicrotome and then incubated with 0.2% ovalbumin for 30 min.at room temperature to block nonspecific sites. Monoclonal antibodyMHS-10 or control IgG₁ was diluted 1:50 in 0.2% ovalbumin and reactedovernight with the sections at 4° C. After exhaustive washing in dropsof PBS, sections were incubated for 2 hours in a 1:25 dilution ofProtein A gold (Janssen Life Sciences, Piscataway, N.J.). Sections werethen washed in PBS and stained for 10 min in 5% uranyl acetate andviewed in a JEOL 100CX electron microscope.

4. Western Blots

Donor sperm were washed in Hams F-10 medium, frozen at -80° C. in thepresence of 5mM benzamidine, 1 mM phenylmethyl-sulfonylfluoride, 2 ug/mlleupeptin, 2 ug/ml pepstatin and thawed and extracted in 1% SDS. Onepart extract was added to one part 2×Laemmli buffer (Laemmli, Nature(Lond), 227:680-85 (1970), incorporated herein by reference) in thepresence or absence of B-mercaptoethanol. Proteins were analyzed by oneand two dimensional electrophoresis according to the procedure ofO'Farrell, J. Biol. Chem., 250:4007-21 (1975), incorporated herein byreference. Electrotransfer followed Towbin et al., Proc. Natl. Acad.Sci. USA, 76:4350-54 (1979), incorporated herein by reference. Thenitrocellulose was blocked in 5% milk in PBS/0.5% Tween-20; incubated inthe MHS-10 mAb (1/1000) in PBS/0.5% Tween-20, 1% milk overnight at 4°C.; goat anti mouse IgG-peroxidase was employed at 1/5,000 dilution.Control IgG₁ monoclonal was also diluted 1/1000. Silver staining ofprotein spots on 2-D gels followed the procedure of Wray et al., Anal.Biochem., 118:197-203 (1981), incorporated herein by reference.

Results

1. SP-10 is a differentiation antigen of spermatogenesis.

SP-10 was found to be expressed at a specific stage of spermdifferentiation in the human testis. Immunohistochemical examination ofparaffin embedded testes (N=3) exposed to the MHS-10 monoclonal antibody(isotype: IgG₁), revealed binding to adluminal spermatids and maturesperm within the seminiferous tubules (FIG. 1A, B). Control sections ofhuman testis incubated with another IgG₁ monoclonal antibody (FIG. 1C)showed no immunoreaction product. Within round spermatids,immunostaining was frequently observed in crescent shaped structures aswell as smaller ovoid granules (FIG. 1B, arrowheads). Groups ofsimilarly stained spermatids which demonstrated either crescent shapedor granular immunoreaction patterns (as in FIG. 1B) were observed incross sections of single seminiferous tubules. This finding isconsistent with previous observations in the human testis that germcells in several stages of differentiation may coexist in any crosssection of a seminiferous tubule. Not all regions of the seminiferousepithelium demonstrated staining, suggesting either a lack of expressionof SP-10 in some stages of spermatogenesis or possible detachment ofsome cells from the seminiferous epithelium in the paraffin embeddedmaterial. Basal spermatogonia, Sertoli cells, spermatocytes, and cellswithin the testicular interstitium showed no immunoreactivity.

2. SP-10 resides within the acrosome of intact sperm.

By immunofluorescence microscopy, SP-10 localized to the human spermhead. Motile, nonpermeabilized sperm (N=1000) which were incubated withthe MHS-10 monoclonal antibody and reacted with a fluorescent secondaryanti-mouse antibody showed no immunofluorescent staining of the sperm(data not shown). This indicated that SP-10 was not present on thesurface plasma membrane of intact sperm at detectable levels. Spermwhich were air dried on slides, fixed with 3% paraformaldehyde,permeabilized with 0.5% Triton X-100 or methanol, and then reacted withthe monoclonal antibody and a fluorescent secondary anti-mouse antibody,stained in a cap-shaped fluorescent pattern. This pattern, similar tothe known morphology of the acrosome, occurred in >90% of sperm in eachsample (FIG. 2A). These results indicated that membrane permeabilizingtreatments rendered SP-10 accessible to antibody binding.

3. Ultrastructural localization indicated SP-10 is associated with theacrosomal membranes.

Fine structural studies were performed to localize SP-10 at higherresolution. Mature ejaculated spermatozoa were fixed in 2%paraformaldehyde and 2% glutaraldehyde, prepared for electronmicroscopy, and immunolabelled on plastic sections with the MHS-10monoclonal antibody and 10 nm gold particles coated with Protein-A. Aconcentration of gold particles was observed over the acrosomalcompartment (FIG. 3). In sections where a portion of the acrosome wassectioned obliquely (as in FIG. 3 at the sperm apex), gold particleswere observed in a bilaminar array. This suggested that in mature,intact sperm, SP-10 is nonuniformly distributed within the acrosome andis associated with the inner and outer acrosomal membranes. Preciseassignment of antigen location at the fine structural level wasdifficult in these preparations because post-fixation in osmiumtetroxide, which defines cellular membranes, was found to destroyantigenicity. However, by comparing nonosmicated, immunolabelledspecimens to osmicated sperm, the position of the acrosomal membraneswas determined to correspond to the electron lucent regions indicated atthe arrowheads in FIG. 3. This led to the conclusion that SP-10 islocated on the races of both inner and outer acrosomal membranesadjacent to the acrosomal matrix in mature, intact, ejaculated sperm.

4. Biochemical characterization

The molecular characteristics of SP-10 were studied by Western blots ofone and two dimensional gels on which sperm homogenates wereelecrophoresed. The pattern of immunoreactive sperm proteins observed onWestern blots of a 10% acrylamide, one dimensional SDS-PAGE gel allowedresolution of at least 14 distinct peptide bands (FIG. 4B), which rangedfrom 18-34 kDa. Sperm homogenates which were treated with SDS and thedisulfide bond reducing agent, B-mercaptoethanol, were compared tohomogenates that were not exposed to the reducing agent (FIG. 4B). Thepattern of immunoreactive peptides was identical whether or notB-mercaptoethanol was present, indicating that reduction of disulfidebonds did not alter the apparent molecular weights of the immunoreactivepeptides.

Silver stain of a sperm homogenate which was electrophoresed on a 2-Dgel showed many protein spots possessing isoelectric points over the pHrange 4.3 to 6.5 (FIG. 5A). The MHS-10 monoclonal antibody immunoreacted(FIG. 5B) with a series of peptide spots which ranged in apparentmolecular weight from 18 to 34 kDa. Immunoreactive peptides withapparent molecular weights from 24-34 kDa had isoelectric points ofapproximately 4.9, while the immunoreactive peptides in the 18 kDa rangewere slightly more basic with pIs from 5.1-5.4.

5. All individuals tested have the SP-10 protein.

FIG. 4B shows that immunoreactive SP-10 from different individuals wasvery similar. The relative intensity of antibody reactivity with any onepeptide band was similar in different individuals, as was the presencein each sperm homogenate of the full complement of 14 distinctimmunoreactive peptide bands. To date, no sperm sample tested, usingeither immunofluorescence or western blots (N=60), has failed to reactwith the MHS-10 monoclonal antibody, indicating that SP-10 is highlyconserved in the human population.

6. SP-10 remains associated with the sperm head following the acrosomereaction.

It is well known that certain constituents of the acrosomal matrixdiffuse from the acrosome during the acrosome reaction, when the outeracrosomal membrane fuses with the sperm plasma membrane. FIG. 2B showsimmunofluorescent staining patterns obtained when the MHS-10 monoclonalantibody was reacted with sperm samples which had been treated with thecalcium ionophore A23187, which induces some of the sperm to undergo theacrosome reaction. Ionophore treated populations contained increasednumbers of sperm showing equatorial bars (FIG. 2B, thin arrowheads) aswell as sperm displaying either faint caps or faint caps and equatorialbars together (FIG. 2B, thick arrowheads).

These light microscopic results indicated that SP-10 remains, in part,associated with the sperm head following the acrosome reaction. Thefaint caps suggested that SP-10 persists on the inner acrosomalmembrane, which is exposed on the sperm head following the acrosomereaction, while the fluorescent equatorial bars indicated retention ofSP-10 in association with the sperm's equatorial segment.

Discussion

The observations that the MHS-10 monoclonal antibody reacts with onlyround spermatids and subsequent stages of spermiogenesis on testissections and localizes within the acrosome at the EM level, coupled tothe report that somatic tissues were non-reactive with the MHS-10monoclonal antibody (Anderson et al., J. Reprod. Immunol., 10:231-57(1987), incorporated herein by reference) together indicate that SP-10may be classified as a "differentiation antigen," i.e., a tissuespecific molecule expressed at a precise stage of human spermatogenesis.MHS-10 immunoreaction product was evident in the seminiferous epitheliumas small ovoid granules adjacent to the nucleus of round spermatids.This staining, indicative of the earliest stage of spermatogenesis atwhich SP-10 was detactable, likely corresponds to the nacent acrosomalvesicle and/or perinuclear Golgi region. The MHS-10 monoclonal antibodythus may offer a useful marker of acrosome development in the human. Oneclinical application of this antibody probe may be in the diagnosis ofthe incidence of immature germ cells (Golgi phase spermatids andsubsequent steps) in semen samples with so-called "round cell syndrome."See Jassim and Festenstein, J. Reprod. Immunol., 11:77-89 (1987),incorporated herein by reference.

The absence of cross reactivity in somatic tissues coupled with itsstage specific expression during germ cell differentiation is alsogermane to the possible utility of SP-10 as a contraceptive vaccineimmunogen. Potential problems of autoimmunity, which would beanticipated if common somatic antigens were utilized as vaccineimmunogens, may not be found with SP-10.

The immunofluorescence evidence indicated that in acrosome intact,membrane permeabilized sperm, SP-10 localized in a cap-shapedimmunofluorescent pattern that appeared to encompass the entire extentof the acrosome in 90% or more sperm from a given donor. There was noevidence that the MHS-10 antibody recognized its cognate antigen on theplasmalemma of living sperm. The report of the WHO workshop (Anderson etal., op. cit. 1987, p. 249) had concluded that the MHS-10 antibody (S20)showed "reactivity . . . with abundant surface antigens on maturesperm." The results reported herein do not agree with this conclusionfor acrosome-intact sperm, obtained from populations containing fewacrosome-reacted sperm.

Our results show that after ionophore induced acrosome reaction, anincrease was noted in the number of sperm displaying fluorescent bars orfluorescent bars together with fainter fluorescent caps. We interpretthe reduced immunofluorescence of the cap (faint cap) to indicate that,following the acrosome reaction SP-10, remains displayed on the spermsurface most likely in association with the inner acrosomal membrane.The retention of immunofluorescence after the acrosome reaction in abelt-like bar likely represents retention of SP-10 within the equatorialsegment. The equatorial bar immunofluorescence, although covering a muchsmaller region than the fluorescent cap, appeared to be of the sameintensity as the complete cap pattern, indicating that the amount ofSP-10 within the equatorial segment is similar before and after theacrosome reaction. The immunofluorescence data was not of sufficientresolution, to determine whether SP-10 remains localized to the innerand/or outer acrosomal membranes and matrix of the equatorial segment orpossibly all of these subdomains following the acrosome reaction, orredistributes to include the plasma membrane overlying the equatorialsegment.

The WHO sponsored multicenter study presented evidence that the MHS-10monoclonal antibody (S20) inhibited sperm egg interactions in thehamster egg penetration test. Our model to explain this resultpostulates that, although sequestered within the limits of the acrosomalmembranes in intact, non-acrosome-reacted sperm, the SP-10 antigen isaccessible to the actions of the MHS-10 antibody following the acrosomereaction.

A common assumption regarding selection of appropriate sperm immunogensfor contraceptive vaccine development is that the target moleculesshould be surface components accessible to humoral or cellular immuneeffectors. Although the intra-acrosomal localization of the SP-10peptides in the mature, non-acrosome-reacted sperm appears at firstglance not to fulfill this caveat, the remodeling of the sperm headmembranes that accompany the acrosome reaction opens the possibilitythat as a class, constituents of the acrosome, although sequestered fromthe immune system in intact sperm, should not be dismissed as candidatesfor contraceptive vaccines without examination of their fate followingthe acrosome reaction.

Studies with guinea pig sperm have provided remarkable evidence thatfull but reversible contraception can be achieved by immunizing femaleanimals with the purified sperm protein, PH-20. See Primakoff et al.,Nature, 335:543-46 (1988), incorporated herein by reference. Thismolecule of 64,000 daltons is present on both the plasma membrane and,following the acrosome reaction, the inner acrosomal membrane of guineapig sperm. See Primakoff et al., J. Cell. Biol., 101:2239 (1985); Myleset al., J. Cell. Biol., 99:1634 (1984); Cowan et al., J. Cell. Biol.,103:1289 (1986); and Primakoff et al., Biol. Reprod., 38:921 (1988), allof which are incorporated herein by reference. PH-20 may play a role insperm binding to the zona pellucida and appears to undergo proteolysisduring the acrosome reaction. Antiserum to the PH-20 protein from guineapig sperm, however, does not cross react with human sperm (Primakoff,personal communication). Although SP-10 and PH 20 appear to be differentmolecules based upon consideration of apparent molecular weight andimmunoreactivity, they share the property of persistence on the spermhead following the acrosome reaction. The remarkable effectiveness of PH20 in eliciting a contraceptive effect in guinea pigs indicates similarcontraceptive potential for SP-10 in humans.

A number of methods, including monoclonal antibody and lectin probes aswell as multiple dye techniques, have been utilized to score theacrosome reaction. See Lee et al., Fertil. Steril., 48:649-58 (1987);Berger et al., Biol. Reprod., 40:525-30 (1989); Cross et al., GameteRes., 15:213-26 (1986); and Wolf et al., Biol. Reprod., 32:1157-62(1985), all of which are incorporated herein by reference. Because theMHS-10 monoclonal antibody is directed to an intra-acrosomal antigenwhich changes from a cap shaped immunofluorescence pattern to a faintcap and/or bar during the acrosome reaction, it may also be usefulclinically in assessing acrosomal status.

We observed a high degree of similarity between individuals in theimmunoreactive forms of SP-10 on Western blots, as well as consistentimmunofluorescent localizations on each individual's sperm, indicatingthat SP-10 is conserved in the human population. This knowledge isessential in choosing a contraceptive vaccine molecule, because it mustbe present on most, if not all sperm, in order for a vaccine to achievethe widest possible effectiveness. The multiple forms of SP-10 peptidesthat are identified by Western blotting may represent post-translationalmodifications, proteolytic processing of the protein within theacrosome, multiple gene products, or several of these possibilitiesacting in concert. The high degree of similarity between individuals onWestern blots suggests that whichever of these alternatives is acting toproduce the polymorphism in antigenic peptides, the mechanisms areoperating similarly in different individuals. The fact that reductiondid not alter the pattern of immunoreactive SP-10 peptides suggests alack of interchain and few or no intrachain disulfide bonds in SP-10.

The electron microscopic localizations in intact, ejaculated human spermindicate that SP-10 is asymmetrically disposed within the acrosomalmatrix, associating in many sperm with the faces of both inner and outeracrosomal membranes adjacent to the acrosomal matrix. Because thepolymorphism of SP-10 is not completely understood at the level of aminoacid sequence and a function for the SP-10 polypeptides has not yet beendetermined, aside from their potential as vaccine immunogens, anunderstanding of the significance of the apparent asymmetry of SP-10 inthe acrosome can only be discussed in a general sense. Knowledge of thespatial organization of various molecules within the acrosomal matrixand acrosomal membranes in intact and acrosome reacted sperm iscurrently in its infancy. The evidence suggests that SP-10 may be acomponent of such an acrosomal "lamina" in human sperm. Moreover, itsasymmetrical distribution in the acrosomal matrix may indicate themolecule contains a hydrophobic domain that directly inserts into theacrosomal membranes.

In summary, by one and two dimensional immunoblots, we showed thatSP-10, extracted from ejaculated human sperm, demonstrated apolymorphism of immunogenic peptides from 18-34 kDa, a pattern which wasconserved from individual to individual and was not altered by reducingagents. The majority of the antigenic peptides possessed isoelectricpoints of approximately 4.9. Immunocytochemistry on testis sectionsindicated SP-10 localized to round spermatids and spermatozoa within theadluminal compartment of the seminiferous epithelium. Immunofluorescenceshowed that SP-10 was not associated with the surface of acrosomeintact, ejaculated sperm. Light and electron microscopicimmunocytochemistry localized SP-10 throughout the acrosome, and EMevidence demonstrated a bilaminar array association with the inneraspect of the outer acrosomal membrane and the outer aspect of the inneracrosomal membrane. Following induction of the acrosome reaction withthe ionophore A232187, SP-10 remained displayed on the sperm head inassociation with the inner acrosomal membrane and equatorial segment.The results indicate that the MHS-10 monoclonal antibody may be utilizedas a marker of acrosome development in the human and as a probe toevaluate acrosome status. The results also support the hypothesis thatinhibition of sperm-egg interaction by anti-SP-10 monoclonal antibodymay occur as a result of antigen exposure following the acrosomereaction.

The testis specificity and stage specific expression of SP-10, itsconservation in the human population, the ability of the MHS-10monoclonal to inhibit fertilization in the hamster egg test, andpreliminary evidence suggesting that SP-10 remains associated with thesperm head following the acrosome reaction, suggests the utility in thishuman sperm molecular as a contraceptive vaccine.

EXAMPLE 8 Identification of Human Acrosomal Antigen SP-10 in Primatesand Pigs

The intra-acrosomal localization of SP-10 has led to speculation as tothe molecule's function. Because the apparent molecular mass of the betaand gamma forms of acrosin (Polakoski and Parrish, J. Biol. Chem.,252:1888-94 (1977), incorporated herein by reference) as well assperminogen (Siegel et al., Biol. Reprod., 36:1063-68 (1988),incorporated herein by reference) overlap with the apparent mass ofSP-10, the question of similarity between SP-10 and these two previouslydescribed intra-acrosomal molecules has arisen. In this study, weutilize purified preparations of pig acrosin and sperminogen (gifts ofKenneth Polakoski) to demonstrate that although SP-10 is present inpigs, it is distinct from acrosin and sperminogen.

Because SP-10 was first defined as a human sperm antigen, theidentification of this molecule in other species will establish a modelfor testing the anti-fertility potential of an SP-10 based contraceptivevaccine. Employing Western and Northern blots, we demonstrate thatprimates and pigs are potential animal models for the study of SP-10.

Materials and Methods

1. Sperm Extracts

Human Sperm. Sperm obtained from donor ejaculates were washed in Ham'sF-10 medium and frozen at -80° C. in the presence of 5 mM benzamidine, 1mM phenylmethylsulfonylfluoride, 2 ug/ml leupeptin, and 2 ug/mlpepstatin. After thawing and extraction in 1% SDS, one part extract wasadded to one part 2×Laemmli buffer (Laemmli, op. cit.) in the presenceor absence of B-mercaptoethanol.

Primate Sperm. Sperm were obtained at the University of WashingtonRegional Primate Research Center from the cauda epididymides of Macacamulatta, Macaca fascicularis, and Papio cynocephalus anubis atsacrifice. The caudae were placed in 10 ml of Human Tubal Fluid (IrvineScientific, Irvine, Calif.), minced into small pieces to allow sperm toescape, and incubated for 15 min. at 37° C., and the resultingsuspension was placed in a 15 ml conical centrifuge tube for 5 min. toallow tissue debris to settle. The supernatant containing sperm wasdecanted and centrifuged at 1,000×g. The pellet was suspended in 500 ulof Laemmli buffer without B-mercaptoethanol and immediately frozen forlater shipping. Upon receipt, samples were diluted 2:1 in 4×Laemmlibuffer with B-mercaptoethanol.

Sperm of Other Species. Rabbit sperm were obtained via an artificialvagina and were a gift from the laboratory of Eugene Oliphant. Bullsperm obtained by electroejaculation were also a gift from thelaboratory of Dr. Oliphant. Rat, pig, and guinea pig sperm were obtainedfrom the cauda epididymides of these species as outlined above with theexception that following centrifugation after collection, these spermpreparations were immediately mediately extracted with 1 ml of 1% SDSand 1% B-mercaptoethanol per 10⁸ sperm.

Protein determinations were performed with the method of Tan, Anal.Biochem., 86:327-331 (1978), incorporated herein by reference. Gels wereloaded with 10 ug sperm extract per lane.

2. Western blots

Sperm extracts were electrophoresed on 10% PAGE-SDS gels andelectrotransferred at 100 mAmps for 12 hours following the procedure ofTowbin et al., op. cit. The nitrocellulose was blocked in 5% milk, 2.5%Tween-20; 1% BSA, 0.5% goat serum and 0.15% gelatin (blocking solution)for 30 min. at room temperature. Nitrocellulose strips were incubated inthe MHS-10 mAb (1/1000 or 1/2000) or control IgG1 (1/1000) in a 1/5dilution of the blocking solution (incubation solution) overnight at 4°C. Following a 3×wash in the incubation solution the secondary antibody,goat anti-mouse IgG-peroxidase, was employed at 1/10,000 dilution on theblots for 2 hours at room temperature. Blots were then washed 3×in PBSand developed with 0.05% DAB and 0.01% H₂ O₂.

3. Northern Blots

Labelled Probe. The open reading frame for SP-10 has been determined byassembling two overlapping cDNAs cloned from human testis by initiallyscreening a testis expression library with the MHS-10 monoclonalantibody. See Example 9. The open reading frame consists of 795nucleotides encoding a protein of 265 amino acids. A fragment consistingof 634 bp of the open reading frame for SP-10, produced due to aninternal EcoR1 site, was nick-translated (Bethesda Research Labs,Rockville, Md.) with P³² dCTP (ICN) and used to probe poly A+ RNA onNorthern blots.

Testis RNA. Human testes were obtained from patients undergoing surgicalorchiectomy for prostate cancer. Baboon (Papio papio, and Papiocynocephalus anubis) and rhesus (Macaca fascicularis) testes wereobtained frozen from the University of Washington Regional PrimateResearch Center. Poly A+ RNA was isolated from these tissues usingOligo(dT)-Cellulose Type 3 (Collaborative Research, Inc., Bedford,Mass.). One microgram of human testes, two ug of human liver andplacenta, and 10 ug of baboon, rhesus, dog, and cat testis poly A+ RNAwere electrophoresed on a 1% formaldehyde-agarose gel according toLehrach et al., Biochem., 16:4743-51 (1977) and Goldberg, Proc. Natl.Acad. Sci. USA, 77:5794-98 (1980), both of which are incorporated hereinby reference. Final membrane washes consisted of 0.1×SSPE, 0.5% SDS at65° C.

Results

Immunoblots of proteins extracted from boar sperm demonstrated that theMHS-10 monoclonal antibody recognized several sperm proteins in thisspecies (FIG. 6, lane 2B). Peptides at 34 kDa, 29 kDa and severalfainter intermediate bands were recognized in both boar and human spermby the MHS-10 monoclonal antibody. Interestingly, the immunoblot of boarsperm protein extracts did not demonstrate several of the peptides below29 kDa which were evident on the immunoblot of the human sperm extract.

Previous studies have reported purification of the boar acrosomalproteins acrosin, Polakoski and Parrish, op. cit., and sperminogen,Siegel et al., op. cit. The kind gift of purified boar sperminogen andboar acrosin by Dr. Kenneth Polakoski allowed us to ask whether theMHS-10 monoclonal antibody would cross react with these previouslydescribed acrosomal matrix constituents. As seen in FIG. 6, the singlebands of purified sperminogen and acrosin lanes 3+4 were unreactive withthe MHS-10 monoclonal antibody, although the boar sperm extract (lane2B) clearly contained immunoreactive proteins. In addition, the purifiedpreparation of acrosin possessed a considerably higher apparentmolecular weight than SP-10. Although the purified preparation ofsperminogen was of similar apparent molecular weight as a majorimmunoreactive peptide of SP-10 at 29 kD, the MHS-10 monoclonal antibodydid not recognize the sperminogen protein band on the Western blot.

Western blotting of sperm extracts of several species, including thebull, rat, and rabbit failed to demonstrate the presence of proteinswhich immunoreacted with monoclonal antibody MHS-10 (FIG. 7). Inaddition to the species shown on in FIG. 7, guinea pig and cat spermextracts were observed to lack reactivity with the MHS-10 monoclonalantibody on Western blots.

Peptides immunoreactive with the MHS-10 monoclonal antibody weredetected on Western blots containing sperm extracts of Papiocynocephalus anubis, Macaca mulatta, and Macaca fascicularis (FIG. 8).Each of these primates showed a polymorphic pattern of immunoreactivitysimilar to the poylmorphic pattern of immunoreactivity observed onextracts of human sperm. However, sperm extracts from each of theseprimates showed immunoreactive peptides of lower apparent mass than inthe human sperm extracts, including a band at approximately 14 kDa.

Northern blots (FIG. 9) which were loaded with poly A+ RNA purified fromtestes of Papio papio, Papio cynocephalus anubis, and Macacafascicularis, demonstrated that these species testes contained a 1.35 kbmRNA which hybridized with the 628 bp SP-10 probe. This 1.35 kb mRNA wasof similar size to human testicular mRNA (FIG. 9). Poly A+ RNA from dogand cat testes did not hybridize with the probe nor did poly A+ RNAobtained from human placenta or liver (FIG. 9).

Discussion

The identification of peptides in pig sperm extracts which wereimmunoreactive with the MHS-10 monoclonal antibody and were of similarapparent molecular weight to human SP-10 indicates that pig spermcontains SP-10. The lack of immunoreactivity of purified preparations ofacrosin or sperminogen with the MHS-10 monoclonal antibody, despite thefact that the pig sperm extract was immunoreactive, indicates adissimilarity between SP-10 and these previously describedintra-acrosomal components. This suggests that the SP-10 protein is anovel intra-acrosomal constituent.

The polymorphic pattern of SP-10 peptides observed on Western blots ofhuman sperm extracts was also observed on sperm peptides obtained frombaboon and rhesus. Why these multiple immunoreactive peptides of varyingmass appear in human and the other primate sperm has not beendetermined. Because the MHS-10 monoclonal antibody was successfullyemployed to screen a lambda gt11 expression library for SP-10, it islikely that the MHS-10 epitope is proteinaceous rather than acarbohydrate. The open reading frame for human SP-10 predicts a proteinof 265 amino acids with a mass of 28.3 kDa. See Example 9. Since twocanonical N-linked glycosylation sites were identified on human SP-10,it is likely that the forms of SP10 with apparent molecular weight above28.3 kDa represent glycosylated SP-10. Each of the Western blots ofprimate sperm showed immunoreactive bands in the upper range of thepattern at approximately 29 kDa. These bands are of approximately themass predicted from the nucleotide sequence without any glycosylation.Like the human sperm extracts, multiple immunoreactive forms below 29kDa were observed in the other primates. This similarity between human,baboon, and macaque SP-10 suggests that the mechanisms responsible forgeneration of the polymorphism of SP-10,be they proteolysis,post-translational modification, multiple gene products, or acombination of these causes, are operating in baboon and macaque spermas well as human sperm.

Pig SP-10, on the other hand, did not demonstrate the multipleimmunoreactive peptides below 29 kDa seen with the extracts of primatesperm. Like the human SP-10, pig sperm immunoblots showed a band atapproximately 34 kDa as well as a major immunoreactive band atapproximately 29 kDa, (approximately the 28.3 kDa mass for the proteinpredicted from the nucleotide sequence). It is unclear at presentwhether this heterogeneity reflects differences in amino acid sequence,post-translational modification, or results from variation in viabilityand proteolysis of the sperm preparations.

Because SP-10 remained associated with the sperm head followingionophore induced acrosome reaction and evidence has been presented thatthe MHS-10 monoclonal antibody inhibited sperm/egg interaction in thehamster egg penetration test, it is possible that SP10 may be aneffective immunogen for inducing antibodies which would interdictfertilization in vivo, provided that sufficient levels of antibody areinduced within the oviduct. The observation that a 1.35 kb mRNA forSP-10 is common to baboons, macaques, and humans provides additionalevidence supporting the similarities observed between humans, baboons,and macaques in immunoreactive SP-10 observed on Western blots. Togetherthese data indicate that macaques and baboons may be appropriate primatemodels for testing the anti-fertility potential of a recombinant SP-10vaccine.

In summary, in the present study, a monoclonal antibody to SP-10(MHS-10) was employed on Western blots to identify immunoreactive SP-10in sperm extracts from baboon (Papio cynocephalus anubis) and twomacaques (Macaca mulatta and Macaca fascicularis). In each of theseprimates, the MHS-10 monoclonal antibody recognized a polymorphicpattern of immunoreactive peptides similar to the human pattern.Immunoreactive SP-10 was also demonstrated in pig sperm. Using purifiedpreparations of the previously described intra-acrosomal moleculesacrosin and sperminogen in the pig, we observed that the MHS-10monoclonal antibody did not react with these proteins, indicating SP-10is distinct from these known acrosomal components. Sperm from severalcommon species, including the rabbit, bull, rat, guinea pig and cat, didnot immunoreact with the MHS-10 monoclonal antibody. Utilizing aradioactive probe spanning 634 nucleotides of the open reading frame forSP-10 on Northern blots of poly A+ RNA obtained from testes of Macacafascicularis, Papio papio, and Papio cynocephalus anubis, a 1.35 kbmessenger RNA of identical size to the mRNA from human testes wasidentified. These results indicate that baboons, macaques, and pigs maybe appropriate models for testing of an SP-10 based contraceptivevaccine.

EXAMPLE 9 Cloning and Sequencing of cDNAs Coding for the HumanIntra-Acrosomal Antigen SP-10

This example describes the characterization of cDNAs coding for thehuman sperm acrosomal protein, SP-10. cDNAs coding for SP-10 wereisolated, sequenced, and the deduced SP-10 amino acid sequence wasanalyzed. This work identified some fundamental characteristics of theSP-10 protein and suggests that alternative splicing of the SP-10 mRNAoccurs. Using the SP-10 cDNAs and the MHS-10 monoclonal antibody, itwill be possible to study the expression of SP-10 during spermatogenesisat both the transcriptional and post-transcriptional levels.Overexpression of SP-10 using the SP-10 cDNAs should also allow us toassess its value as a contraceptive vaccine immunogen. Assessingimmunogenicity of several SP-10 peptides using recombinant methods isalso made possible by having cloned and sequenced the SP-10 cDNAs.

Materials and Methods

1. Isolation and Analysis of cDNA Clones

The MHS-10 monoclonal antibody was used to probe a human testis lambdagt11 expression library. The library was a gift from Jose Millan. SeeMillan et al., Proc. Natl. Acad. Sci. USA, 84:5311-15 (1987),incorporated herein by reference. The library was plated at a density of5×10⁴ plaque-forming units per 150 mm Petri dish with E. coli Y1090 ashost bacterium. After growth at 42° C. and induction with isopropyl-B-Dthiogalactoside, the nitrocellulose filters were preincubated with 5%milk and 5% goat serum and screened with a 1:1000 dilution of MHS-10monoclonal antibody (isotype IgG1). Bound MHS-10 was detected by use ofa goat anti-mouse IgG coupled to horseradish peroxidase (JacksonImmunoResearch Laboratories). A putative clone was identified uponscreening 50,000 pfu from the expression library. This phage was plaquepurified using MHS-10 and showed no reactivity to other IgGl monoclonalsor to the goat anti-mouse IgG. This clone contained a cDNA insert of 214base pairs (bp), designated SP-10-214, which was nicktranslated(Bethesda Research Labs ) with p³² dCTP (ICN) and used to reprobe thegt11 library to identify additional clones using the procedure of Bentonand Davis, Science, 196:180-182 (1977), incorporated herein byreference. Five additional clones were identified.

Three plaques homologous to the 214 bp clone were purified and the phageDNAs isolated. These phage DNAs were digested with EcoR1 and run on a 1%agarose gel. The EcoR1 digestion produced cDNA insert bands ofapproximately 650 bp and 400 bp for all three isolates. Northern blotsperformed using either the 650 bp or the 400 bp insert of cDNA SP-10-5as a probe gave identical results (data not shown). The 650 bp and 400bp inserts for all three phage isolates were isolated and subcloned intopGEM3Zf (ProMega). The cDNA designated SP-10-5 is a composite of thecDNA inserts contained in the plasmids pGEM-SP-5-650 and pGEM-SP5-400.The cDNAs designated SP-10-8 and SP-10-10 are also composites of theirrespective 650 bp and 400 bp cDNAs fragments. Nested deletions were madefrom each end of the cDNA fragment in pGEM-SP-5-650 and pGEM-SP-5-400using the Erase-a-Base System (ProMega), and both strands of each insertwere sequenced using a Sequenase sequencing kit (US Biochemicals).Nested deletions were made from one end of the 650 bp SP-10-10 cDNAfragment in pGEM-SP-10-650 and one strand was sequenced. The 400 bpSP-10-10 fragment in pGEM-SP-10-650 was sequenced by priming from bothends. The entire open reading frame for SP-10 is a composite constructedfrom the SP-10-5 and SP-10-10 cDNAs.

2. Homology Analysis

Homology searches of the Genbank, National Biomedical ResearchFoundation (NBRF) protein and Swiss Protein Library databases wereperformed using the Pearson and Lipman FASTA and LFASTA programs. SeePearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-48 (1988),incorporated herein by reference. Comparisons were run with ktups of 1and 2.

3. RNA Isolation and Northern Blots

Human testes were obtained from elective orchiectomies for prostatecarcinoma from patients untreated with steroids and frozen in liquidnitrogen. The tissue was then ground to a powder on dry ice and the RNAisolated using guanidine isothiocyanate followed by CsCl centrifugation.Chirgwin et al., Biochemistry, 18:5294-99 (1979), incorporated herein byreference. Poly(A)+ RNA was isolated using oligo(dT)-cellulose(Collaborative Research) as described by Bantle et al., Anal. Biochem.,72:413-427 (1976), incorporated herein by reference.

One microgram of human testes poly (A)+ RNA and 2 ug of human placentaland liver poly (A)+ RNAs were electrophoresed on a 1%formaldehyde-agarose gel. See Lehrach et al., Biochem., 16:4743-51(1977), and Goldberg, Proc. Natl. Acad. Sci. USA, 77:5794-98 (1980),both of which are incorporated herein by reference. The RNA was blottedto Biotrace membrane (Gelman), and its integrity was judged bybackshadowing the 18S and 28S ribosomal RNA bands with U.V. light. Themembranes were prehybridized (50% formamide, 1% milk, 5×SSPE, 1% SDS and100 ug/ml salmon sperm DNA) and then hybridized with a P³² labelled 634bp fragment containing part of the coding region from SP-10-5 (bps67-695). Final membrane washes consisted of 0.2×SSPE, 0.5% SDS at 65° C.

Results

1. Characterization of the SP-10 cDNAs

A human testis cDNA expression library was screened using the MHS-10monoclonal antibody. The cDNA insert of one MHS-10 reactive plaque waspurified, sequenced, and found to be 214 bp in length. This insert wasthen used as a probe to isolate 3 larger cDNAs designated as SP-10-5,SP-10-8, and SP-10-10. A 634 bp fragment of cDNA SP-10-5 was used toprobe northern blots containing poly (A)+ RNA from human testes, liver,and placenta (FIG. 10). One band at 1.35 kb was present in the testesRNA but not in either the liver or placental RNA lanes.

Sequence analysis revealed that cDNAs SP-10-5 and SP-10-10 overlappedextensively (FIG. 11A). By combining the sequences for SP-10-5 andSP-10-10, a partial cDNA of 1117 bp with an open reading frame of 795bases, 265 amino acids, was identified for the SP-10 protein. With theexception of an in-frame deletion of 57 bp (19 amino acids) in SP-10-10,the remaining overlapping sequences for SP-10-5 and SP-10-10 wereidentical. The 5' and 3' ends of the SP-10-8 sequence were identical tothe SP-10-5 and SP-10-10 cDNA sequences where they overlapped, but likeSP-10-5 did not have the 57 bp deletion present in the SP-10-10sequence.

The sequence analysis also identified a consensus polyadenylationsequence at position 1094, 236 bp 3' of the TAG termination codon, and aputative eukaryotic mRNA degradation sequence 71 bp 3' of the stopcodon. The 5' sequence (CCAG) that flanked the initiator methionine wassimilar to a consensus sequence found 5' to most eukaryotic startcodons.

The amino acid sequence for SP-10 deduced from the cDNA sequencepredicted a protein of 28.3 kD. Three different repeating amino acidmotifs were identified (FIG. 11A). The first motif (Ser, Gly, Glu, Gln,(Pro or Ala)) occurred 7 times. There were two additional variants ofthe first repeat, (Val, Gly, Glu, Gln, Pro) and (Ser, Asp, Glu, Gln,Pro) which differed by only one amino acid. The second motif (Ser, Glu,His, (Gly or Ala), Ser) was repeated 3 times, while the third motif(Ser, Gly, Glu, His) was repeated 4 times. These three motifs comprised76 of the 108 amino acids between amino acids 66 and 174.

A hydrophobicity plot of the SP-10 amino acid sequence (FIG. 11B) showeda hydrophobic amino terminus characteristic of a signal peptide. Thecentral portion of the protein that contained the repeated motifs hadseveral hydrophilic domains while the carboxy terminus was quitehydrophobic. Two canonical N-linked glycosylation sequences(Asp-X-Ser(Thr)) existed at amino acids 48 and 258, while a stretch ofserines and threonines that began at amino acid 80 suggested possibleO-linked glycosylation sites. The sequence, (Ser-(Asp or Glu)-X-X-Pro),which occurred at residue 140, has also been suggested as a possibletarget site for O-linked glycosylation (Gerry Hart, personalcommunication).

2. Homology Searches

The entire SP-10 cDNA and amino acid sequences were compared to theGenbank, NBRF, and Swiss sequence banks using the library searchprograms Fasta and tFasta at ktups of both 1 and 2. Neither the SP-10nucleic acid or amino acid sequences showed any homology with thesequences in the banks. The 3 repeated amino acid motifs were alsocompared to the same sequence banks. While several proteins contained asingle copy of one motif, none contained multiple copies of any motif.

Discussion

We have described the cloning and initial characterization of cDNAscoding for the human sperm acrosomal protein SP-10. Sequence analysis ofthe SP-10 cDNAs revealed several interesting features of the SP-10protein. A hydrophobicity plot generated from the deduced amino acidsequence showed SP-10 contained a strongly hydrophobic amino terminuscharacteristic of a signal peptide. Furthermore, when the amino terminal20 amino acids were analyzed individually for charge and hydrophobicity,they conformed well to the characteristics of a signal peptide. A signalpeptide would be required to transport the SP-10 protein through themembrane of the endoplasmic reticulum and into the Golgi vesicles thatcoalesce to form the developing acrosome.

Following the signal sequence, a central peptide core exists containingseveral hydrophilic domains comprised almost entirely of the threerepeating peptide motifs. It should be noted that because of the 19amino acid deletion, cDNA SP-10-10 is missing a single copy of two ofthe motifs. The role these unique repeats play in the functioning ofSP-10 is still unclear since no proteins were found in the Genbank,NBRF, and Swiss sequence banks that contained multiple copies of themotifs.

Analysis of the cDNA sequences revealed 2 potential N-linkedglycosylation sites and possible O-linked glycosylation sites.Carbohydrates at these positions could account for the difference insize between the 34 kd SP-10 species observed in Western blots and the28.3 kd peptide (26 kd after removal of the signal peptide) calculatedfrom the deduced amino acid sequence. This size discrepancy wasexpected, since other acrosomal proteins have been shown to beglycosylated, including acrosin of the rabbit, boar, and goat. Human andboar proacrosin, for example, migrated at approximately 55 kd by SDSPAGE but were synthesized from mRNAs that coded for peptides of onlyabout 45 kd.

The size heterogeneity previously observed for the SP-10 protein isapparent in FIG. 12. Differing degrees of glycosylation could accountfor some of the heterogeneity on Western blots. The internal deletionwithin the SP-10-10 cDNA suggested that differential splicing of theSP-10 transcript might also account for some of the heterogeneity. TheSP-10-10 mRNA with a 19 amino acid deletion would code for a protein 2kd smaller than that produced by the SP-10-5 mRNA. However, it isunlikely that alternative splicing was the major cause of theheterogeneity, since the SP-10 transcript was a relatively discrete bandon Northern blots. Proteolysis of the SP-10 protein during spermmaturation and storage probably also contributed to its sizeheterogeneity. The fact that all three SP-10 cDNAs isolated to dateshared identical 3' untranslated sequences where they overlappedsuggested that multiple SP-10 genomic genes were probably notresponsible for the heterogeneity.

The production of a MHS-10 immunoreactive fusion protein from theoriginal 214 bp cDNA has localized the MHS-10 epitope to a 71 amino acidpeptide of the SP-10 protein. The amino terminal 34 amino acids of this71 amino acid peptide were comprised entirely of two of the three typesof hydrophilic motifs, while the carboxy terminal 37 amino acids werequite hydrophobic (FIG. 11B). Since MHS-10 was very likely generated toa hydrophilic region of the SP-10 peptide, one or more of the motifs inthis 34 amino acid stretch probably comprises or contributes to theMHS-10 epitope.

The availability of the cDNAs has allowed us to generate large amountsof SP-10 as a fusion protein. The recombinant antigen will allow us totest two hypotheses: 1) that SP-10 may be effective as a contraceptivevaccine immunogen; and 2) that sera from persons with anti-spermantibodies may recognize recombinant SP-10. If the latter proves to bethe case, immobilized recombinant SP-10 may serve as a useful targetantigen for measuring anti-sperm antibodies.

In summary, cDNAs coding for the intra-acrosomal protein SP-10 werecloned and characterized as a first step in understanding the expressionof this antigen during spermatogenesis. Three overlapping SP-10 specificcDNAs were isolated from a human testes cDNA expression library. ThesecDNAs hybridized to a 1.35 kb mRNA which was present in human testes butwas not found in liver or placenta. Complete sequencing of these cDNAs,designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117 bp sequencecontaining a 265 amino acid coding region for the SP-10 protein.Hydrophobicity plots generated from the deduced amino acid sequenceshowed a very hydrophobic amino terminus characteristic of a signalpeptide. Sequence data showed that three different amino acid repeatsoccurred a total of 16 times in the central third of the SP-10 protein.Interestingly, cDNA SP-10-10 has a internal 57 bp (19aa) in-framedeletion which is not present in SP-10-5, suggesting that alternativesplicing generates more than one SP-10 mRNA. The SP-10 protein appearsto be a unique acrosomal protein based on previous immunohistologicaldata and on the observation that SP-10 cDNA sequences did not show anysignificant homology to other sequences found in the Genbank, NBRF, orSwiss sequence banks.

EXAMPLE 10 Preparation of Antigen by Transformed Microorganisms

The SP-10-5A cDNA insert will be excised from pGE-SP-10-5 with KPN I andSST I. The ends will be blunted using Mung Bean Nuclease and NcoIlinkers attached with T4 DNA ligase. (The SP-10-5 cDNA contains nointernal NcoI sites.) The linkers will then be cleaved with NcoI and thecDNA separated from unligated linkers by agarose gel electrophoresis andelectroelution of the fragment. The cDNA will then be ligated into theNcoI site of the E. coli expression vector pKK233-2 (Pharmacia). Thisvector contains an IPTG (isopropyl-B-D-thiogalacto-pyranoside, U.S.Biochemicals Corp.) inducible promotor and the lacZ ribosome bindingsite 5' to the cDNA insertion site and a consensus transcriptiontermination sequence 3' to the cDNA insertion site. The linkers ligatedto the cDNA will be a mixture of 3 NcoI linkers, each containing an AUGstart codon in one of the three reading frames. The cDNAs ligated intopKK233-2 will be transformed into E coli. The resulting colonies will betransferred to nitrocellulose and the filter placed onto an agar platecontaining 2 mM IPTG for 4 hrs at 37° C. to induce production of theSP-10-5A protein. The filter is then incubated at 100° C. in 5% sodiumdodecyl sulfate (SDS), dried, and incubated with MHS-10 MAB andhorseradish peroxidase-labeled goat anti-mouse antisera. Coloniesshowing a positive reaction with the SP-10 MAB will be isolated and 1 mlovernight cultures grown in the presence of 2 mM IPIG. The recombinantE. coli will be collected by centrifugation and lysed in 4×proteinloading buffer (4% SDS, 20 mM TRIS (pH 8.0), 0.5M 2-mercaptoethanol, 20%glycerol). These samples will be boiled, subjected to 1D SDS PAGEaccording to Laemli, Nature (Lond) 227:680 (1970), incorporated hereinby reference, and Western blotted with the MHS-10 MAB and horseradishperoxidase labeled goat anti-mouse antisera. Those colonies showing anMHS-10 reactive band of approximately 27 kD (SP-10-5 cDNA codes for 266amino acids) will be used to start large cultures for isolation of therecombinant SP-10-5 protein. Recombinant E. coli collected from thelarge preps will be lysed to release the SP-10-5 protein. Aftercentrifugation to remove the cellular debris, the SP-10-5 protein willbe purified using ion-exchange chromatography, MHS-10 MAB affinitychromatography, and preparative electrophoresis.

Expression in pGEX

The pGEX system produces a "pure" (non-fusion) recombinant protein whichwe intend to use as a vaccine immunogen both alone and as a conjugatewith other proteins which enhance the immune system. We havere-engineered the SP.-10-5 cDNA SP-10-5 into the plasmid expressionvectors pGEX -2T and pGEX-3X. Smith and Johnson, Gene 67:31-40 (1988),incorporated herein by reference. We have observed overexpression ofrecombinant SP-10. These constructs give a fusion polypeptide with thecarboxyl terminus of the Schistosoma japonicum glutathione S-transferase protein. Smith et al., PNAS 83:8703-8707 (1986),incorporated herein by reference. Most fusion proteins produced in thissystem are soluble in aqueous solutions and can be purified from crudebacterial lysates under non denaturing conditions by affinitychromatography on immobilized glutathion. Using batch wash proceduresseveral fusion proteins can be purified in parallel in under two hourswith yields of up to 15 mg protein/liter of culture. Pure SP-10 isprepared by cleavage from the glutathione S-transferase carrier bydigestion with site specific proteases such as thrombin (for pGEX-2T)and blood coagulation factor X_(a) (for pGEX-3X). After digestion, thecarrier and any uncleaved fusion protein are removed by absorption onglutathione agarose.

EXAMPLE 11 Testing Prototype Recombinant Vaccine for Immunogenicity inRabbits

Materials and Methods

1. Generation of SP-10 Rabbit Polyclonal Antisera

A 634 bp SP-10-5 EcoR1 fragment (bps 67-701, 202aa) and the original 214bp SP-10 cDNA (bps 487-701, 71aa) that had EcoR1 ends were inserted intothe E. coli expression vectors pWR590 and pWR591 respectively. See Guoet al., GENE, 29:251-254 (1984), incorporated herein by reference. TheSP-10/B-galactosidase fusion protein that resulted from the 634 bpinsertion was isolated according to Guo et al. and subjected to SDSPAGE. The SP-10/B-galactosidase fusion protein band was excised from thegel, frozen, ground to a powder, and resuspended in PBS.

Two rabbits were injected subcutaneously with equal volumes of the gelslurry and Freund's Complete Adjuvant (Gibco) and then were injectedtwice more with the gel slurry in Freund's Incomplete Adjuvant at twoweek intervals. Rabbits were bled, and the blood was processed for IgGusing ammonium sulfate precipitation.

2. Western Blots and Immunofluorescence with Polyclonal AntiseraGenerated to a Prototype Recombinant Vaccine

Donor sperm were washed in Ham's F-10 medium and frozen at -80° C. inwater. After being thawed and vortexed, the sample was centrifuged at10,000×g for 30 seconds, and one part supernatant was added to one part2×Laemmli buffer (Laemmli, op. cit.) with B-mercaptoethanol. Theproteins were subjected to SDS PAGE, transferred to nitrocellulose(Towbin et al., op. cit.), blocked in 5% milk in PBS/0.5% Tween-20, andincubated in a 1:1000 dilution of MHS-10, null ascities, SP-10 rabbitpolyclonal, or rabbit preimmune sera in PBS/0.5% Tween 20, 1% milk for 2hrs at room temperature. Goat anti-mouse (Jackson ImmunoResearch Labs)or goat anti-rabbit (HyClone) IgG-horseradish peroxidase was used at1:5000 dilution, and reaction product was developed with 0.05%diaminobenzidine with 0.015% hydrogen peroxide. All washes betweenantibody incubations were done with PBS/Tween/1% milk.

For immunofluoresence studies, sperm were washed as described above,resuspended in PBS, and air dryed on slides. The slides were submergedin 3% paraformaldehyde for 30 min and methanol for 20 min to fix andpermeabilize the sperm. They were preincubated in 10% goat serum in PBSfor 15 min, and then incubated in monoclonal antibody MHS10, nullascities, rabbit SP-10 polyclonal antisera, or rabbit preimmune antiseraat a 1:500 dilution for 1 hr at room temperature in a humidity chamber.The slides were washed 5×in PBS and incubated in fluorescein labelledgoat anti-mouse antisera (Jackson ImmunoResearch) (for MHS-10 and nullascities) or fluorescein labelled goat anti-rabbit antisera (HyClone)(for SP-10 polyclonal antisera and preimmune antisera) at 1:500dilutions at room temperature for 1 hr in a humidity chamber. The slideswere then washed 5×in PBS, mounted with 90% glycerol in 25 mM Tris (pH8.0) and viewed under a Zeiss phase microscope equipped withepifluoresence.

Results

1. Western Blots and Immunofluorescent Localization

A 634 bp SP-10-5 fragment (bps 68-701, 210aa) and the original 214 bpSP-10 cDNA (bps 487-701, 71aa) were inserted onto the E. coli expressionvector pWR590 and expressed as B-galactosidase fusion proteins. The twoconstructs, identified as pWRSP-210 and pWRSP-71, produced fusionproteins that reacted specifically with MHS-10 on Western blots (datanot shown). The pWRSP-210 fusion protein was used to generate polyclonalantisera in two rabbits. The antisera was used to probe Western blotscontaining SDS solubilized sperm extracts (FIG. 12). (The antiseraproduced by the two rabbits reacted identically on Western blots and inthe immunofluorescent localization study; therefore only the data fromrabbit #1 was shown here.) The polyclonal antisera reacted with the sameseries of bands (17-34 kd) on the human sperm extract lane as did themAB MHS-10. No cross-reactivity was visible between the SP-10 polyclonalantisera and other non-SP-10 sperm proteins. The rabbit preimmune serashowed no reactivity with any sperm protein bands.

Paraformaldehyde fixed human sperm were reacted first with the SP-10polyclonal antisera and then with a fluorescein labelled goatanti-rabbit secondary antibody. Only a cap on the head of the sperm,similar in morphology to the acrosome, showed any reactivity with thepolyclonal antisera (FIG. 13A). This cap shaped immunofluorescent imagewas identical to that stained with monoclonal antibody MHS-10 (FIG.13C). The preimmune antisera and null ascities showed no staining of thesperm at all (FIGS. 13B and 13D).

Discussion

The observations that the polyclonal antisera raised to theSP-10/B-galactosidase fusion protein: 1) reacted with the identicalseries of peptides on Western blots as did monoclonal antibody MHS-10and; 2) showed precise immunofluorescent staining of the sperm acrosomalcap, provide two mutually supporting proofs that the isolated cDNAs codefor the SP-10 protein. Had the SP-10 cDNAs coded for a non-SP-10 proteinthat only shared the MHS-10 epitope, the Western blot andimmunofluorescence data would likely not have been identical for MHS-10and the SP-10 polyclonal antisera. The innoculated rabbits showed noapparent ill effect of receiving the SP-10 recombinant vaccine. Thissuggests the recombinant vaccine may prove to be safe and efficacious.The fact that the recombinant vaccine evoked a polyclonal response whichrecognized the native SP-10 provides further proof that the recombinantvaccine will be efficacious.

A recombinant SP-10 fusion protein was produced in an E. coli expressionvector and used to generate a polyclonal antisera. This antisera stainedthe acrosomal cap in-situ and reacted with a similar set of peptides onWestern blots as did a monoclonal antibody to SP-10.

EXAMPLE 12 Chromosomal Location

Genomic blots containing mouse/human cell hybrid DNAs were hybridizedwith the 5' 634 bp portion of SP-10-5. Table I shows the hybridspositive when screened for the SP-10 gene and indicates which complementof chromosomes were contained in these hybrids.

This table is compiled from 33 cell hybrids involving 16 unrelated humancell lines and 4 mouse cell lines. See Shows, et al., Advances in HumanGenetics, Volume 12, Eds. H. Harris and K. Hirschhorn, (Plenum Press,New York and London), 1982, pp. 341-452; Shows, et al., Somat. Cell Mol.Gen., 10:315-318 (1984); and Shows, et al., Cytogenet. Cell Genet. 21:99-104 (1978), all of which are incorporated herein by reference. Thehybrids were characterized by karotypic analysis and by mapped enzymemarkers. See Shows, TB. 1983, Isozymes: Current Topics in Biological andMedical Research, Volume 10, pp. 323-339, Eds. M. C. Rattazzi, J. G.Scandalios, and G. S. Whitt, Alan R. Liss, New York, incorporated hereinby reference. The "t" in the table indicates a chromosome translocationfor a particular chromosome, but no intact chromosome is present. (Seeunder Translocations).

The DNA probe for the DNA probe SP-10 was hybridized to Southern Blotscontaining EcoRI digested DNA from the human-mouse hybrids listed in thetable. The scoring for the probe SP-10 was determined by the presence(+) or absence (-) of human bands in the in the hybrids on the blots.Concordant hybrids have either retained of lost the human bands togetherwith a specific human chromosome. Discordant hybrids have eitherretained the human bands, but not a specific chromosome or the reverse.Percent discordancy indicates the degree of discordant segregation for amarker and a chromosome. A 0% discordancy is the basis for chromosomeassignment.

The DNA probe for SP-10 mapped to human chromosome 11 by somatic cellhybrids. The hybrid XER-7 with the 11/X translocation: 11p12 or11p11→11gter::Xq11→Xqter and the hybrid EXR-5CSAZ with the X/11translocation: Xpter→Xq22::11q13→11qter would localize the SP-10 to theP12→q13 region of human chromosome 11.

Chromosome 11 gave a concordancy of 31 and a discordancy of X.Chromosome 16 gave the next highest concordancy and discordancy figuresof 23 and 13 respectively. This data indicates that the genomic gene forSP-10 is located on chromosome #11, probably in the area of the 11q2band.

EXAMPLE 13 Differential Diagnosis of Immature Germ Cells in SemenUtilizing Monoclonal Antibody MHS-10

Human semen contains, in addition to spermatozoa, a population of roundnucleated cells predominantly composed of germ cells, originating fromthe testis, and inflammatory cells (leukocytes). Although, in fertileindividuals, round cells represent less than 5% of the total number ofcells in semen, they are increased in cases of infertility associatedwith infection or hormonal alterations of normal spermatogenesis. Germcells found in semen include spermatids and spermatocytes. Thedifferentiation between the different stages of sperm precursors andleukocytes by light microscopy of semen smears using conventionalstaining techniques has been unreliable, due to morphologicalsimilarities in size, and requires a highly trained eye for accuratediagnosis. Round spermatids and spermatocytes could be mistaken forlymphocytes, while non-separated spermatids sharing a common cytoplasmcould be mistaken for polymorphic nuclear leucocytes.

Anti-leukocyte monoclonal antibodies have recently been employed inimmunocytochemical techniques to define leukocytes and theirsubpopulations in semen smears. Identification of sperm precursors usingpolyclonal antibodies raised against human germ cells and sperm has alsobeen attempted using immunofluorescence assays followed by toluidineblue staining, but evaluation was difficult and necessitated thesubsequent use of electron microscopy for positive identification. SeeJassim and Festenstein, J. Reprod. Immunol., 11:77 (1987), incorporatedherein by reference.

This example shows a simple and reliable method for the differentialanalysis of immature germ cells in semen smears using a monoclonalantibody (mAb) probe, MHS-10 (IgG₁). This antibody recognizes a humansperm protein, (SP-10), which has been immunocytochemically localized byelectron microscopy to Golgi phase spermatids and all subsequent phasesof spermigenesis.

In this example, the MHS-10 antibody was used to histochemically stainsemen smears using a standard immunoperoxidase technique. To evaluatepotential cross reactivity with leukocytes, anti-HLe-1 (a pan-antihumanleukocyte mAb probe) was also used. The results indicate that round cellpopulations staining with anti-SP-10 did not stain with anti-HLe-1.Spermatids at varying stages of acrosome development could be detectedby the anti-SP-10 monoclonal antibody. The use of this antibody probealso allows for the rapid identification of various types ofmorphologically abnormal germ cells in semen smears.

Materials and Methods

1. Semen samples

Semen samples were obtained from 34 subjects. Seven of these were fromfertile men defined by having fathered at least one child and having norecent history of venereal infection. Three were from severelyoligospermic patients (<10×10° sperm/ml ejaculate). Five were fromazoospermic patients. Eight were from polyspermic patients (>25°×100°sperm/ml ejaculate). Six were from patients defined as having increasedround cells in their semen and five were from vasectomized patients. Theinfertile patients were from Brigham and Women's Hospital, Boston, Mass.Routine semen analysis was performed as described in Hill, et al.,Fertil. Steril., 47:460 (1987), incorporated herein by reference.

2. Preparation of semen smears

Liquefied semen was centrifuged at 600×g for 10 minutes. The seminalplasma was aspirated and the cellular pellet washed twice with phosphatebuffered saline (PBS: 0.01M, pH 7.2). The final pellet was resuspendedin PBS to approximately 10⁷ cells/ml and 5 ul of this suspension wasapplied to each spot of 8-spot Teflon-coated microscope slides (RobozSurgical, Washington, D.C.). The slides were dried and fixed in acetonefor 10 minutes and frozen at -70° C.

3. Monoclonal antibodies

MHS-10 cell line (IgG₁) was subcloned two times and grown as ascitestumors. Balb/c mice (Charles River, Boston, Mass.) were primed with twoi.p. injections of 0.5 ml sterile Pristane(2,6,10,14-tetramethylpentadecane: Sigma Chemical Co., St. Louis, Mo.)at 2-week intervals. One week following the second injection, 10⁷hybridoma cells were injected i.p. in 0.5 ml serum-free, sterileRPMI-1640 medium (Gibco, Grand Island, N.Y.). The ascites fluid wascollected and cleared of cellular debris by centrifugation (1,000×g) andstored at -60° C. until needed. Anti-HIe-1 was purchased from BectonDickinson, Mountain View, Calif.

4. Immunohistologic staining of sperm and round cells

Semen smears were immunohistochemically stained using theStreptavidinbiotin-peroxidase system (SBP) (Histostain SP-kit, ZymedLaboratories South San Francisco, Calif.) as described in Wolf andAnderson, Fertil. Steril., 49:497 (1988), incorporated herein byreference. After saturation of non-specific binding sites withnon-immune rabbit serum for 10 minutes, 10 ul of mAb was incubated onindividual spots of the slides for 30 minutes at 37° C. Biotinylatedsecondary antibody was then added (10 ul) for 10 minutes, followed by 10ul of streptavidin peroxidase conjugate for 5 minutes. Immunoreactionproduct was developed with the chromogen aminoethylcarbazole in thepresence of the substrate hydrogen peroxide for 5 minutes at 37° C. Thesmears were counterstained with hematoxylin and mounted by a aqueousmounting medium.

Each specimen slide had one spot to which 10 ul PBS was added, insteadof primary antibody as control. In addition, they each had one spot towhich MHS-10 ascites fluid (diluted 1/1000) was added, one spot to whichanti-HLe-1 (diluted 1/20) was added, one spot to which a mixture ofMHS-10 (diluted 1/250), and anti-HLe-1 (diluted 1/20) was added. Alldilutions were made with PBS.

Evaluation of immunoperoxidase stained smears was made by DifferentialInterference Contrast microscopy using a Leitz 100/1.32 DIC objective ona Leitz Ortholux microscope equipped with a Leitz Vario Orthomat camera.Photographs were made with Ektachrome 160 Tungsten film.

Results

1. Immunostaining of semen smears

Within the semen smears, immature germ cells which had been sloughed atvarious stages of formation in the testis could be detected with theMHS-10 antibody probe to the intra-acrosomal antigen SP-10. Examples ofMHS-10 positive germs cells are assembled according to stages ofacrosome development (FIG. 14B-F). FIG. 14B depicts an early stagespermatid prior to the onset of acrosome formation. FIG. 14C showsimmunohistochemical staining of a developing spermatid containing anovoid MHS-10 positive granule lying adjacent to the nucleus. This figurelikely represents an early acrosomal granule in a Golgi phase spermatid.FIG. 14D shows a somewhat larger immunostained acrosomal granule in theGolgi phase of spermiogenesis as well as an incomplete flagellum.

A spermatid displaying an uncondensed, open nucleus and animmunoreactive crescent is apparent in FIG. 14E. This represents a moreadvanced stage of acrosomogenesis, likely a cap phase spermatid. In thisfigure, the flattened acrosome is in a position proximal to theimplantation site of the flagellum, a feature characteristic of earlyspermatid differentiation during which the flagellar anlage is implantedat the nucleus. Mature sperm, abundant in normal specimens (FIG. 14F)showed immunostained acrosomes, enveloping the condensed nucleus and ina position distal to the flagellum, typical of completedacrosomogenesis.

Abberant germ cell morphologies indicative of defective cytokinesis wereobserved using the MHS-10 mAb. (FIG. 15A-E). These included binucleatedspermatids (FIG. 15A), spermatids containing two acrosomal granuleswithin the same cell (FIG. 15B), and sperm containing two condensednuclei enveloped by two acroscmes within a single sperm head (FIG. 15Carrows). Images such as those seen in FIGS. 15D-E were interpreted asrepresenting intact intracellular bridges where daughter spermatidsremained attached and were apparently sloughed off as a cohort of cells,displaying asynchronous development. Of the four attached cells seen inFIG. 15D, one germ cell was staged at the Golgi phase of spermiogenesis(arrows points to the acrosomal vesicle) and three others were staged atthe Golgi phase of development.

Other abnormal sperm phenotypes were also observed in semen stainedusing the monoclonal antibody MHS-10. Biflagellated tails were observedin germ cells showing immunoreactive cap phase acrosomes and uncondensednuclei (FIG. 15F). Sperm displaying uncondensed nuclei withmicroacrosomes (FIG. 15G) as well as cap phase spermatids lackingflagella (FIG. 15H) were also observed.

In some cases, reactive acrosomal remnants were observed withinpleomorphic structures resembling fragments of sperm heads containingnuclear material (FIG. 15I). Examples were also observed (FIG. 15J-K) ofa peripheral cuff of MHS-10 positive reaction product beneath thelimiting membrane of the cell.

In the negative control experiments in which PBS was used instead of themAb in semen smears, there was no red-brown immunoreaction product dueto immunoperoxidase and only the blue hematoxylin counterstain of spermand round cells was observed (data not shown). In semen smears that hadbeen treated with the anti-HIe-1 monoclonal antibody, only theleukocytes reacted histochemically, as evidenced by the red-brown stainof AEC. Mature sperm and spermatids did not cross react with theanti-HLe-1 antibody and remained blue (FIG. 15L).

Discussion

At present, it is difficult during semen analysis to distinguishleukocytes from sperm precursors using conventional light microscopicmethods. The general category of "round cells" often serves todistinguish all other cell types present in semen from sperm. Theconventional staining techniques used in the past such as thePapanicolaou stain or a combination of Leishman's blood stain andBryan's sperm stain impart only general morphological information on thecellular components of the ejaculates. Overlap in sizes of the "roundcells" is one cause of difficulties in definitive diagnosis. Granularleukocytes range in size from 9-14 mm while nongranular leukocytes rangefrom 6-12 uM. The average size of a spermatid is 5-6 um in diameter.Analysis of semen smears containing mixtures of germ cells andleukocytes using conventional stains is time-consuming and requirescareful inspection of individual round cells, with distinction betweenthe lymphocyte and immature germ cells being particularly problematic.

In the present study, we made use of a unique monoclonal antibody and astandard immunoperoxidase technique employing the chromogen AEC toeasily visualize the target round cells. The MHS-10 antibody inconjunction with this method localized sperm precursors beginning withthe Golgi and subsequent phases of spermiogensis. Cryosections of humantestis stained using this mAb have shown it to target a developmentalstage-specific antigen, (SP-10), appearing on adluminal germ cells andmature sperm but not on spermatogonia. The intra-acrosomal locus of theSP-10 antigen as well as its testis specificity have been established.The antigen is absent on cells from adrenals, colon, brain, skin,tonsils, lungs, liver, kidney, ovary, and endometrium and does not reactwith serum and peripheral blood leukocytes. Conversely, the leukocyteantibody HLe-1, as was confirmed in the present study, does not reactwith germ cells or mature sperm.

The application of the MHS-10 mAb probe to semen smears allows thedetection of sperm as well as immature germ cells that had beenpreviously sloughed off from the testis at various stages ofspermiogenesis. Jassim and Festenstein, op. cit., have used a mouseanti-human sperm polyclonal antibody to visualize round cells in semen.Immunological identification of the various stages of germ celldifferentiation, however, was not possible at the light microscopy levelin their study, since the antibody used was not acrosome-specific andreacted with the cell surface of all germ cells. Their studies at theelectron microscopy level, however, showed the presence of germ cells atvarious stages of differentiation. These authors have also demonstratedthe presence of germ cells with abnormal morphology (such as binucleatedcells) in semen. Electron microscopy was the method of choice by whichthis could be demonstrated. The latter method, although affording highresolution morphological data, is time-consuming and would have littleapplication in clinical laboratory settings. The MHS-10 monoclonalantibody immunoreagent offers advantages of constant affinity and class,uniformity and availability in virtually unlimited supply, givingdiagnosis of the MHS-10 positive subset of spermatids a standard ofuniformity and reproducibility that was previously difficult to achievewith polyclonal immunoreagents.

Immunohistochemical staining of semen smears using MHS-10 allowed theidentification of clusters of daughter spermatids connected byintracellular bridges. Such clusters could be indicative of failure ofcytokinesis. Partial failures of the testis in the disjunction processmay account for the presence of multinucleated sperm in the ejaculate.When groups of germ cells connected by intracellular bridges are notsubjected to added disruptive forces in the testis, the constrictionsbetween them gradually disappear and a spherical multinucleated mass isformed that contains as many nuclei as were conjoined in the originalcluster of cells. Although the exact stage and mechanism(s) of theseparation of spermatids into individual spermatozoa is not known, theMHS-10 probe allows for rigid quantitation of such cell associations.

Spermatids found joined together by intracellular bridges are morelikely to occur in a syncytial relationship, and in normalspermatogenesis, coordination of development is achieved by uniformdistribution of chemical factors controlling differentiation. Thus, itis not clear why cohorts of coupled spermatids stained with MHS-10 wereobserved to be at different stages of acrosome formation (FIG. 15D-E).Dym and Fawcett, Biol. Reprod., 4:195 (1971), incorporated herein byreference, reported the occurrence of multiple transverse cisternae inthe bridges joining dividing spermatogonia of the ram and rat. Thepresence of these membranous structures temporarily interrupted thecontinuity between the cell bodies of the conjoined cells, thusresulting in slight asynchrony of their cellular development. Themembrane-limited cisternae persisted for only a short time afterreconstitution of the nuclei of the daughter cells, although septatebridges were also observed in association with postkaryokineticspermatid nuclei. Observations such as these have been limited to testisof experimental animals and have never been reported in human semen. TheMHS-10 antibody probe has thus allowed documentation of the occurrenceof asynchromous cohorts of spermatids in human semen for the first time.

Our understanding of all the factors controlling cytokinesis in thetestis and the mechanism by which germ cells, either in clusters, orindividually, are shed (spermiation) into the ejaculate is poorlyunderstood. Further studies on the significance of germ cells in semenneed to be undertaken. The MHS-10 probe may prove very useful indefining the relative proportion of specific subsets of germ cellsprematurely shed at specific stages of their development, acategorization which could help clarify different types of testicularpathology underlying the cause of "round cell" syndrome. Soderstrom andSuominen, Arch. Pathol. Lab. Med., 104:476 (1980), incorporated hereinby reference, have demonstrated by electron microscopy studies ontesticular biopsies that meiotic arrest is associated with anaccumulation of pachytene spermatocytes and a lack of spermatids in theseminiferous tubules. This increase would be likely to be reflected inthe semen of such patients such that the use of a stage-specific mAbreacting with pachytene spermatocytes would be a useful marker for therapid identification of patients with meiotic arrest. In the same way,cellular accumulation in semen of a specific stage of spermatiddevelopment identified by MHS-10 could shed light upon a pathologycausing subfertility or infertility of men with "round cell" syndrome.

In summary, acetone dried smears from washed human semen containingsignificant numbers of round cells were probed with mAb MHS-10.Monoclonal antibody labelled cells were visualized by a standardstreptavidin-biotin immunoperoxidase method using a light microscope.The MHS-10 mAb immunoreacted with mature sperm and with a subset ofround cells diagnosed as developing spermatids which has been sloughedoff from the testis at varying stages of acrosome formation. To rule outpossible cross-reactivity of the mAb with leukocytes in semen, aleukocyte surface marker (anti-HLe-1) was used in conjunction withMHS-10. Round cell populations staining with MHS-10 did not stain withanti-HLe-1. The MHS-10 mAb provides a unique immunoreagent fordifferential diagnosis of a subset of immature germ cells during semenanalysis. The mAb MHS-10 is thus a promising probe for theidentification and quantitation of immature germ cells in human semen.

Summary of Experimental Results for Examples 1-13

The above examples show that the human sperm protein, SP-10, is adifferentiation antigen which is detected in round spermatids at theGolgi phase and subsequent steps of spermiogenesis. SP-10 localizeswithin the nacent acrosomal vesicle of spermatids, is an intra-acrosomalprotein in mature sperm, and appears to be testis-specific. The proteinremains associated with the equatorial segment and/or inner acrosomalmembranes of ionophore induced acrosome reacted sperm.

These observations have led to the suggestion that SP-10 and its cognatemonoclonal antibody MHS-10 provide a useful marker/probe system for: a)diagnosing immature germ cells in semen; and b) scoring the acrosomereaction. Furthermore, SP-10 has been designated a "primary vaccinecandidate" by the World Health Organization Taskforce on ContraceptiveVaccines, due to its tissue specificity and evidence that the MHS-10monoclonal antibody inhibits fertilization in the hamster eggpenetration test.

On immunoblots of human sperm extracts, polymorphism of immunoreactiveSP-10 peptides are observed to range from 18 to 34 kDa. This pattern ofimmunoreactivity with the monoclonal antibody MHS-10 has been shown tobe conserved from individual to individual and to be unaffected byreducing agents. Western blots of 2-D gels have shown that the antigenicpeptides of 24-34 kD have a pI of 4.9 whereas the peptides ofapproximately 18 kD are more basic, with pI's ranging from 5.1-5.4.

cDNAs coding for the intra-acrosomal protein SP-10 were cloned andcharacterized. Three overlapping SP-10 specific cDNAs were isolated froma human testis cDNA expression library. These cDNAs hybridized to a 1.35kb mRNA which was present in human testes but was not found in liver orplacenta. Complete sequencing of these cDNAs produced an 1117 bpsequence containing a 265 amino acid coding region for the SP-10protein. SP-10 has a predicted molecular weight of 28.3 kD.Hydrophobicity plots generated from the deduced amino acid sequenceshowed a very hydrophobic amino terminus characteristic of a signalpeptide. SP-10 appears to be a unique acrosomal protein based onprevious immunohistological data and on the observation that SP-10 cDNAsequences did not show any significant homology to other sequences foundin three databases.

A recombinant SP-10 fusion protein was produced in an E. coli expressionvector and this prototype recombinant vaccine was used to generate apolyclonal antisera in rabbits. This rabbit antisera stained theacrosomal cap in-situ and reacted with a similar set of peptides onWestern blots as did a monoclonal antibody to SP-10. The rabbits did notappear to suffer from the vaccine. These results show that a recombinantSP-10 vaccine is capable of evoking in mammals an immune response whichrecognizes the native human sperm SP-10.

EXAMPLE 14 Cloning and Sequencing of Primate SP-10

Introduction

SP-10 is an acrosomal protein that is first detected in the developingacrosomes of round spermatids in the human testis. 1,2!. In mature,ejaculated sperm, SP-10 is specifically localized to the intra-acrosomalcompartment and appears to be associated with the acrosomal membranes2!. Analyzed by 1 and 2-dimensional SDS-PAGE and Western immunoblots,SP-10 presents as a series of polymorphic peptides ranging from 18 to 34KDa, the majority of which have isoelectric points of 4.9 2!.

SP-10 has been designated a "primary contraceptive vaccine candidate" bya WHO taskforce on contraceptive vaccines on the basis of severalcharacteristics 3!. First, current tissue specificity data suggest thatSP-10 is specific to maturing germ cells within the testis 1,4,5!. Suchtissue specificity reduces the likelihood of autoimmune disease arisingin females who are administered an SP-10 vaccine. Second, SP-10 has beendetected in the sperm of all human males tested to date (N>200), andthus appears to be conserved among males 2!. Third, SP-10 remainsassociated with the sperm head after the acrosome reaction 2!. Finally,a monoclonal antibody to SP-10 (MHS-10) was shown to inhibit human spermpenetration in the hamster egg penetration assay 3!. Additionalpreliminary data have shown human IVF to be inhibited by a monoclonalantibody which reacts with a molecule considered to be SP-10 6!.

Human SP-10 has been cloned and sequenced and its amino acid sequencededuced from cDNAs 4!. Two alternatively spliced forms of SP-10 wereisolated which encode proteins of 246 and 265 amino acids. The two cDNAsencode identical proteins except for a 19 amino acid deletion in thecentral portion of the smaller protein 4!.

Northern analysis has shown that SP-10 mRNA is present in both baboon(Papio papio) and cynomolgus monkey (Macaca fasicularis) testes, andWestern blots of sperm extracts indicate that baboon and macaque SP-10display multiple immunoreactive forms similar to human SP-10 5!.Electron microscopic immunolocalization of SP-10 in baboon testis usingcolloidal gold and monoclonal antibody to human SP-10 (MHS-10) has shownbaboon SP-10 to be present within the acrosomal region of the developingsperm 7!. Thus, both baboons and macaques are possible candidates forfertility trials utilizing SP-10 as the vaccine immunogen.

In the present study, to further evaluate baboons and macaques as modelsfor testing recombinant human SP-10 as a contraceptive vaccineimmunogen, cDNAs for baboon and macaque SP-10 were cloned and sequenced.A comparison of the deduced SP-10 amino acid sequences of human, baboon,and macaque reveals that the protein exhibits a high degree of homologyin these primate species. The regions of highest homology provideimportant information for the rational design of recombinant SP-10contraceptive vaccine formulations. The results also demonstrate thatSP-10 mRNAs are alternatively spliced in several species.

Materials and Methods

1. Construction of baboon and macaque cDNA testis libraries

Testis libraries were constructed using Stratagene's Lambda Zap-cDNAsynthesis kit and protocols (Stratagene, La Jolla, Calif.). Briefly,baboon (Papio papio) and macaque (Macaca fasicularis) testes werehomogenized in 4M guanidinium thiocyante, 25 mM sodium citrate, 0.5%sarcosyl, and 0.1M 2-mercaptoethanol. Total RNA was isolated via cesiumchloride centrifugation as described by Maniatis 8!, and poly(A+) RNAwas purified by oligo-d(T) chromatography 9!. cDNA was synthesizedstarting with 5 ug of poly(A+) mRNA using Moloney-Murine Leukemia Virus(MMLV) reverse transcriptase and an oligo-d(T) linker-primer. Secondstrand synthesis proceeded with the addition of RNase H, freshnucleotides, and DNA Polymerase I. The cDNA termini were blunted and EcoRI adaptors were ligated onto both ends of the cDNA followed by Xho Idigestion. The cDNAs were then directionally ligated into pBluescriptvector arms predigested with Eco RI and Xho I. The plasmid was packagedinto lambda coat proteins using Stratagene's Zap-cDNA Gigapack II goldcloning kit and protocol. The library was amplified, aliquoted, andstored in 7% DMSO at -80° C.

2. Library screening

Libraries were titered using XL1-Blue host cells and plated on 150 mm×15mm NZY plates at 50,000 plaques/plate as described in the Stratageneprotocols. Three hundred thousand plaques were screened by the followingmethod. Duplicate nylon filters (Micron Separations Inc., Westboro,Mass.) were placed on the plates and marked with ink. The DNA wasdenatured in 1.5M NaCl/0.5M NaOH, neutralized in 1.5M NaCl/0.5MTris-HCl, and rinsed in 0.2M Tris-HCl/2×SSC for 2, 5, and 2 min.,respectively. The DNA was cross-linked to the filters with UVirradiation and then prehybridized overnight in a solution containing50% formamide, 5×SSC (1×=150 mM NaCl, 15 mM sodium citrate), 1% milk, 1%SDS, and carrier DNA (0.1 mg/ml) at 42° C. The filters were thenhybridized overnight at 42° C. in fresh 50% formamide solutioncontaining 10 ng/ml ³² p-dCTP (ICN Biomedicals, Costa Mesa, Calif.)labeled 634 base pair human SP-10 cDNA probe 4!. This probe encoded theamino terminal 2/3 of the SP-10 protein. The filters were washed 3×20min. in 0.2×SSC/0.5% SDS at 65° C. and placed on Kodak XAR-5 filmovernight at -80° C. with an intensifying screen. Positive plaques wereselected by coring the agar and the phage liberated in 1 ml of SM buffer(100 mM NaCl, 8 mM magnesium sulfate, 50 mM Tris-Cl, pH 7.5, 0.01%gelatin) with 20 ul chloroform at 25° C. for 3 hours. Liberated phagestocks were titered, plated, and rescreened when individual plaques wereisolated.

3. DNA sequencing

Plasmid DNA from positive clones was isolated by alkaline lysis andpurified through PEG precipitation as described by Kraft et al. 10!. Thedouble stranded DNA template was sequenced from both directions usingSequenase (United States Biochemical Corp., Cleveland, Ohio) and thedideoxy chain termination method with T3 and T7 primers. Sequencingreactions were heated at 95° C. for 3 min., separated on a 6% acrylamidesequencing gel and apparatus (International Biotechnologies, Inc., NewHaven, Conn.) at 1500V, fixed for 1 hour (850 ml dH₂ O, 100 milmethanol, 50 ml acetic acid), dried, and placed on Kodak XAR-5 film atroom temperature.

4. Nested deletions

Nested deletions of full length clones were made from both directionsusing the Erase-a-Base (Promega, Madison, Wis.) kit and protocols.Briefly, plasmid DNA was double digested leaving a 5'overhang towardsthe insert and a 3'overhang towards the vector. Exonuclease III digestedthe DNA at a rate of 400 bases/min. at 35° C. from the 5'overhang.Aliquots were taken at 30 second intervals producing 200 base deletionsthrough the entire insert. The deletion termini were blunted, ligated,and transformed into XLI-Blue host cells. The deletions weresubsequently sequenced as previously described.

5. PCR reactions

Two micrograms of poly(A+) RNA (baboon and macaque) was reversetranscribed using MMLV and oligo-d(T) as a primer as described in theStratagene protocols. 10% (5 ul) of the resulting cDNAs were amplifiedby PCR (Perkin-Elmer-Cetus, Norwalk, Conn.) for 40 cycles: 94° C. for 1min./37° C. for 2 min./72° C. for 3 min. 9!. The baboon and macaqueSP-10 specific primers 5' d(GGGGATCCATGAACATGTTTCTCTTACTAATG) and 5'd(GGCCTAGGCTAGATCTTATTACAGAAACATTG) mapped to the initiation andtermination of translation, respectively. Aliquots of the resulting PCRproducts were separated on a 5% polyacrylamide gel at 200V for 3 hoursand then the gel was stained with ethidium bromide in 1×TBE 89 mM Trisbase, 89 mM boric acid, 2 mM EDTA).

6. Primer extension

A synthetic 31mer oligonucleotide 5' d(GGGGATCCATTAGTAAGAGAAACATGTTCAT)complementary to nucleotide positions 71 to 93 in the baboon and macaquecDNAs with a 5' Bam H1 linker and a GG clamp was used as an extensionprimer. Two micrograms of Poly(A+) RNA (baboon and macaque) wascoprecipitated with 2.5 ng of ³² p-dCTP 5' end-labeled primer. Thepellet was resuspended in 2.5 ul sterile dH₂ O, and then 2.5 ul 2M NaCl,0.2M Pipes, 5 mM EDTA were added and mixed. The mixture was annealed at55° C. for 2 hours. Reverse transcription was carried out in a 50 ulvolume by adding 33 ul sdH₂ O, 5 ul 10×buffer (250 mM Tris-base, 80 mMMgCl₂, 4 mM DTT), and 2 ul MMLV (18 U/ul) for 1 hour at 37° C. Sampleswere ethanol precipitated in 3 volumes at -20° C. overnight, resuspendedin formamide dye, and denatured at 90° C. for 3 minutes. The sequenceladder was produced using the synthetic oligonucleotide described aboveas the primer and a baboon SP-10 cDNA as the template in a sequencingreaction. The primer extension reactions and the sequencing ladder wereheated at 95° C. for 3 min., separated on a 6% acrylamide sequencing gelat 1500V for 3 hours, fixed for 1 hour (850 ml dH₂ O, 100 ml methanol,50 ml acetic acid), dried, and placed on Kodak XAR-5 film with anintensifying screen at -80° C.

Results

1. Cloning and sequence analysis of baboon and macaque SP-10 cDNAs

The baboon and macaque testis cDNA libraries were screened with a 634base pair (bp) human SP-10 cDNA probe 4! and positive clones wereidentified in each. These clones were isolated and both strands of thethree largest cDNAs from each library were sequenced. Sequencingrevealed two distinct full length SP-10 cDNAs in each species which were1.1 Kb and 1.2 Kb in length. The 1.1 Kb SP-10 cDNAs from baboon andmacaque each contained an open reading frame of 753 bp, while the 1.2 KbSP-10) cDNAs from baboon and macaque each contained an open readingframe of 855 bp. Within the same species, the nucleotide sequence of the1.1 Kb and 1.2 Kb cDNA were identical, with the exception of a 102 bpdeletion in the 1.1 Kb cDNA. The cDNAs sequenced from both species alsocontained up to 70 nucleotides of the 5' untranslated region and 267nucleotides of the 3' untranslated region.

The 1.2 Kb SP-10 cDNA sequences from baboon and macaque were analyzedand compared to each other and then compared to the human SP-10 sequenceutilizing the EuGene sequence analysis program (Baylor College ofMedicine, Houston, Tex.) (FIG. 16). The 1.2 Kb SP-10 cDNAs from baboonand macaque shared an overall 98% homology. No nucleotide substitutionswere observed within the 5' untranslated region, 7 were found within theopen reading frame, and 13 were noted within the 3' untranslated region.

Within the 5' untranslated region in the baboon and macaque, a stretchof 70 nucleotides demonstrated a 100% homology. This region containedthe sequence AAACCGAG located adjacent to the translation start site.This was similar but not identical to the conserved motif AAATCAAA whichhas been found next to many eukaryotic start codons 11!.

The open reading frames in the 1.2 Kb SP-10 cDNAs of baboon and macaquehad a 99% homology over 855 nucleotides. The initiation codons (ATG)began at nucleotide 71 in both species and the termination codons(TAG-Macaque; TAA-Baboon) occurred at nucleotide 927. Within the openreading frame, both species exhibited alternative splicing, whichresulted in the formation of two distinct cDNAs: 1.1 Kb and 1.2 Kb. The1.1 Kb baboon and macaque SP-10 cDNAs each had identical internaldeletions which were 102 nucleotides in length. The flanking sequence ofthese deletions encoded the 5'GTG-GAG 3' consensus splice sequencecharacteristic of an intron.

Following the open reading frame, the 3' untranslated region showed a95% homology between baboons and macaques over 267 nucleotides extendingfrom the stop codon through the beginning of the poly A tail. Therelatively lower homology in this region resulted from sequencedivergence between baboons and macaques over a span of 25 nucleotides(1172-1197) following the polyadenylation consensus sequence. In bothprimates, a putative eukaryotic mRNA degradation sequence ATTTA waslocated at nucleotides 970-974 and the polyadenylation sequence AATAAA11! was located at nucleotides 1166-1171 in the 1.2 Kb cDNAs.

2. Sequence comparison of nonhuman primate and human SP-10 cDNAs

Like the baboon and macaque SP-10 cDNAs, human SP-10 has shown twoalternatively spliced mRNAs 4!. Sequence comparison between the largestalternatively spliced human and baboon SP-10 cDNAs revealed an overallsequence homology of 89% (FIG. 16). The human SP-10 cDNA contained 51nucleotides in the 5' untranslated region and within this region sharedan 88% homology with the baboon. The open reading frame of the human andbaboon cDNAs shared a 79% homology. Within the open reading frame, thehuman SP-10 cDNA contained 60 fewer nucleotides than the 1.2 Kb babooncDNA resulting in a lowered regional homology. However, the sequencefollowing the deletion and extending through the end of the open readingframe exhibited a local homology of 98% between the human and baboonover 245 nucleotides. The 3' untranslated region in the human cDNAcontained 250 nucleotides and shared a 96% homology to the baboon cDNA.This region of the human SP-10 cDNA contained the putative mRNAdegradation sequence ATTTA and the polyadenylation sequence AATAAA atthe same positions as contained in the baboon sequence. Also, the humanSP-10 cDNA contained a single base deletion at nucleotide 1165immediately 5' to the polyadenylation sequence.

3. Primer extension analysis of baboon and macaque mRNAs

Primer extension analysis indicated a single, major transcriptionalstart site and 3 possible minor start sites in the baboon and macaque(FIG. 17). The major start site was located at nucleotide 4 of thebaboon and macaque cDNAs, 67 nucleotides 5' to the ATG codon. Minorstart sites existed 69, 95, and 100 nucleotides 5' to the ATG codon.Both the major and the minor transcriptional start sites mapped toprecisely the same nucleotides in baboons, macaques, and humans 12!.

4. PCR analysis of SP-10 mRNA alternative splicing

Alternative splicing of human SP-10 mRNA was previously demonstrated tooccur within the coding region of the protein 4!. PCR experiments onbaboon and macaque reverse transcribed testis poly A RNA confirmed theexistence of alternatively spliced SP-10 mRNAs and that this splicingoccurred within the coding region. Primate SP-10 specificoligonucleotide primers were used in the PCR such that amplified PCRproducts contained the entire open reading frame. The amplificationproducts for both primates were separated on an acrylamide gel andstained with ethidium bromide (FIG. 18). The result was two bands perlane that migrated at approximately 850 and 750 nucleotides whichcorresponded precisely to the size of the open reading frame of the 1.2Kb and 1.1 Kb cDNAs, respectively. Within the same species, the bandswere present in about equal ratios which concurred with our resultsobtained from the cDNA library screening which showed the frequency ofthe individual transcripts was about 1:1.

5. Amino acid analysis of baboon and macaque SP-10

The 1.2 Kb baboon and macaque SP-10 cDNAs contained identical openreading frames of 855 nucleotides that encoded proteins of 285 aminoacids (FIG. 19). These proteins shared a greater than 98% (285/289)homology differing by only four conserved amino acid substitutions andhad deduced molecular weights of 30.1 kDa. The alternatively spliced 1.1Kb cDNAs contained open reading frames of 753 nucleotides that encodedproteins of 251 amino acids with deduced molecular weights of 26.8 kDa.The alternative splicing in both species resulted in an SP-10 variantwith an internal deletion of 34 amino acids. Both baboon and macaqueSP-10 contained two canonical N-linked glycosylation sites (N-X-S/T) atresidues 48 and 278 13, 14!.

Hydropathy analysis of the deduced amino acid sequences of baboon andmacaque SP-10 indicated that the proteins could be subdivided into asignal peptide of approximately 18 residues 15! and 2 distinct regions:a hydrophilic region that included the amino terminal two thirds or theprotein and a hydrophobic region that included the carboxy terminal theprotein.

The amino terminal two thirds of baboon and macaque SP-10 contained 189amino acids which were 98% (185/189) homologous. This region containedthe alternative splice sites and was characterized by 3 major repeatmotifs consisting of 5 residues: (V/S, G, E, Q, P/S), (P/L/S, G, E, H,A/L), and (S, E, H, G/A, S). The pentapeptides occurred 9, 7, and 3times, respectively. 14 of these repeats were arranged into 3 adjacentlarger repeat motifs each containing 25 amino acids. The splicecontained the last 3.6 pentapeptide repeats along with 16 otherresidues.

The carboxyl terminal one third of the protein (baboon and macaque)contained 78 residues and was 100% (78/78) homologous. This region wasrelatively hydrophobic and contained 10 cysteine residues.

6. Amino acid comparison of nonhuman primate SP-10, human SP-10 andMSA-63

The amino acid sequence comparison between baboon and human SP-10revealed an overall homology of 85% (242/285). This included 242 exactmatches, 20 conserved substitutions, 3 nonconserved substitutions, and20 residues for which there was no match due to a deletion in the humansequence. The % homology was determined by dividing the number of exactamino acid matches by the number of total possible matches.

Comparisons of the mouse intra-acrosomal antigen, MSA-63 171!, toprimate SP-10 amino acid sequences showed a 53% (151/285) homology withbaboon SP-10 and a 60% (158/265) homology with human SP-10. The twocanonical N-linked glycosylation sites (N-X-S/T) at residues 48 and 278are conserved in the nonhuman primate and human SP-10 sequences, whileonly the second site at residue 278 was present in MSA-63 13,14!.

Comparison of the hydropathy plots of the deduced amino acid sequencesof baboon and human SP-10 and mouse MSA-63 indicated that these proteinscontained similar regions. All 3 proteins contained a hydrophobic leadersequence characteristic signal peptide 15!, a distinct hydrophilic aminoterminal region, and a more hydrophobic carboxyl region.

The hydrophilic amino terminal two thirds of baboon and human SP-10shared a 78% (148/189) homology, while baboon SP-10 and MSA-63 sharedonly a 39% (73/189) homology in this region. Human SP-10 contained thesame 3 pentapeptide repeats 4! as in baboon SP-10. Eleven of thesepentapeptide repeats formed 2.5 larger repeat domains of 25 residues:the third 25 residue repeat was truncated. MSA-63 did not contain therepeat motifs to the same extent having only 2 repeats of (S, G, E, Q,P/S) and 3 repeats of (S, G/T, E, H, T/T).

The hydrophobic carboxyl region (78 residues) exhibited the greatestdegree of interspecies conservation. There was a 99% (77/78) and 86%(67/78) homology between baboon SP-10 compared to human SP-10 andMSA-63, respectively. Of particular interest in the hydrophobic carboxylterminal region are 10 cysteine residues that were absolutely conservedin human, baboon, macaque, and mouse.

7. Homology searches

Nonhuman primate SP-10 cDNAs and amino acid sequences were compared toavailable sequences in Genbank using the fasta search program in Eugene.Only a mouse sperm antigen (MSA-63) exhibited homology to SP-10 above10%.

Discussion

The cloning and characterization of the acrosomal protein SP-10 in thebaboon and macaque were undertaken in anticipation of fertility trialsusing human SP-10 as a contraceptive vaccinogen in female baboons and/ormacaques. The appropriateness of testing the human immunogen in nonhumanprimates depends on a high level of homology between human and nonhumanprimate SP-10. For example, other studies that have utilized thebeta-subunit of human chorionic gonadotropin (beta-hCG) as acontraceptive vaccinogen encountered difficulties because of a limitedcross-reactivity between antibodies raised against hCG and othernonhuman primate CC's due to differences in the beta-subunit of thehormone; the least cross-reactive were baboon CG, macaque CG, andmarmoset CG 16!. In the present work, we postulate that significanthomology between human SP-10 and the nonhuman primate models wouldpredict higher cross-reactive antibody titers and possibly lowerfertility. The high degree of homology between human SP-10 and bothbaboon and macaque SP-10 indicates these two species are appropriatemodels for testing a human SP-10 vaccine.

The SP-10 mRNAs of human, baboon, and macaque show alternative splicing.All sequence comparisons were made between the largest spliced forms ofeach species. The 1.2 Kb baboon and macaque SP-10 cDNAs were 98%homologous and each showed an 89% homology to the human SP-10 cDNA. The1.2 Kb nonhuman primate clones have 60 additional nucleotides within theopen reading frame not found in the larger human SP-10 clone. These 60nucleotides correspond to the first 60 nucleotides of the 102 nucleotidealternative splice in baboon and macaque SP-10. Genomic sequencing hasshown that this entire region of human SP-10 is encoded by a single exon(nucleotides 122-685) 12! indicating that these 60 nucleotides have beendeleted from the human genome. The absence of these sequences frombaboon and macaque alternatively spliced SP-10 suggests that the aminoacids encoded by this sequence may not be essential to the functions ofSP-10.

The alternative splice sites in both human and nonhuman primate SP-10cDNAs each began near the C-terminal end of the repeat domain andextended eleven amino acids beyond the repeat motifs to terminate at thesame amino acid. The ratio of the two alternatively spliced transcriptsin baboons and macaques was about 1:1, however, the ratio may varybetween individuals. In contrast, in humans the larger transcript is thepredominant form 12!. The function of the alternative splicing isunclear at this time, but it probably contributes to the differences inmass for human and nonhuman SP-10 peptides on Western blots 5!, byforming SP-10 peptides differing by 19 (human) and 34 (baboon andmacaque) amino acids 2!.

The deduced amino acid sequences of baboon and macaque SP-10 exhibited a98% homology to each other. Each of these proteins exhibited an 85%homology to human SP-10, while a 53% homology exists between baboonSP-10 and MSA-63, and a 60% homology exists between human SP-10 andMSA-63. These homologies are similar to interspecies homologies of thetestis specific gene product, LDH-C, another possible contraceptivevaccine candidate described by Goldberg 17!. A 98% homology was foundbetween human and baboon LDH-C E. Goldberg, Personal communication! anda 73% homology between human and mouse LDH-C 18!.

Human, baboon, and macaque SP-10 and MSA-63 all demonstrated an aminoterminus characteristic of a leader peptide consisting of 18 amino acids15!. This signal sequence could traffic the protein into the endoplasmicreticulum and through the Golgi apparatus ultimately to coalesce in thedeveloping acrosomal vesicle in the early spermatid, whereimmunoreactive SP-10 has been first observed 1!.

Another intriguing region of human, baboon, and macaque SP-10 and MSA-63is an internal hydrophilic region that constitutes 50% of the proteinand contains 3 major repeat motifs consisting of 5 residues: (V/S, G, E,Q, P/S), (P/L/S, G, E, H, A/L), and (S, E, H, G/A, S). In baboons,macaques, and humans, most of these repeats can be grouped into threelarger adjacent repeat motifs each consisting of 25 residues. The repeatmotifs contain several probable endoproteolytic cleavage sites thoughtto be responsible for the characteristic polymorphic pattern of primateSP-10 peptides when SP-10 is extracted in its native form from theacrosome 5,19!. These cleavages result in SP-10 peptides which containthe repeat region as their amino terminal portion. MSA-63 does notcontain the repeat motifs to the same extent having only 2 repeats of(S, G, E, Q, P/S) and 3 repeats of (S, G/T, E, H, T/L). However, MSA-63does contain four of the postulated proteolytic cleavage sites alongwith several closely related motifs that differ by only one or tworesidues as described by Herr et al. 19!. The biological role of thehydrophilic region of repeat motifs is not known. It may be speculatedthat the hydrophilicity may mediate interaction of SP-10 with componentsof the acrosomal matrix.

The C-terminal third (78 residues) of the primate SP-10 proteins as wellas MSA-63 demonstrated the highest interspecies conservation. The highdegree of conservation in this region implies a similar function in allspecies. Lee et. al. have suggested that MSA-63 is associated with actinwhich may serve to anchor the protein to the acrosomal membranes 20!. Insuch an association, the hydrophobic region could anchor SP-10 to themembranes as SP-10 has no classical membrane spanning domain 13, 14!. Insupport of a membrane association, electron microscopicimmunolocalization of human SP-10 showed that in certain immumogoldlabeled EM sections of human sperm, the distribution of gold particleslines up adjacent to the membranes of the acrosomal compartment 5!.Additionally, within this region there are 10 cysteine residues that arecompletely conserved in all 4 species. However, Western blots of reducedand non-reduced human SP-10 are similar suggesting a lack of disulfidebonds 2!.

The 5' untranslated regions of the baboon, macaque, and human SP-10cDNAs contained up to 100 bases. Primer extension analysis of baboon andmacaque SP-10 mRNAs indicated that transcription was initiated 100, 95,69, and 67 bases upstream of the ATG codon in all three species. Themajor start site was located 67 bases upstream while three minor startsites were located 100, 95, and 69 bases upstream. During cDNA libraryscreening with the human SP-10 probe, cDNAs for baboon and macaque SP-10were obtained that included sequence 5' to the major transcriptionalstart site. These cDNAs probably represent transcription initiation atone of the minor upstream start sites. Human SP-10 mRNA yieldedidentically sized extension products indicating that both the major andminor transcriptional start sites are genuine 12!.

In summary, characterization of primate SP-10 cDNAs was undertaken inthe anticipation of fertility trials using human SP-10 as acontraceptive vaccinogen in female baboons. We have demonstrated thathuman and baboon SP-10 are sufficiently homologous such that antibodiesraised against human SP-10 in baboons may be predicted to recognizeSP-10 on baboon or macaque sperm. The carboxyl terminus of SP-10demonstrated the highest interspecies conservation, and we suggest anyvaccine which incorporates SP-10 as an immunogen should incorporate thisregion, as it likely contains functionally essential epitopes.

EXAMPLE 15 Purification of SP-10

Human SP-10 and the polymorphic polypeptides comprising it were purifiedby a 3-step process involving affinity chromatography followed byreverse phase HPLC and preparative SDS-PAGE.

Protein A Column Preparation: Three grams of protein A sepharose CL-4B(Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) were swollen in 50ml of phosphate buffered saline (PBS), pH 8.0, for 20 minutes. Theswollen gel was poured into a 10 ml disposable syringe fitted with ateflon support and a 3-way stopcock. The column was washed with 50 ml ofPBS pH 8.0, followed by 0.01M citrate buffer, 0.15M NaCl, pH 3.0, andfinally reequilibrated with PBS pH 8.0. When not in use it was stored inPBS with 0.2% sodium azide at 4° C.

Antibody Precipitation: Saturated ammonium sulfate (6.4 ml, 4° C.) wasadded dropwise with stirring to 8 ml MHS-10 ascites fluid and stirred at4° C. for 3 hours. The precipitate was pelleted by centrifugation at10,000 rpm for 10 minutes in a Sorvall RC-5B with a SS-34 rotor. Thesupernatant was discarded and the pellet was resuspended in 5 ml PBS,and then dialyzed against PBS at pH 8.0 hrs with 4 changes of PBS.

Purification of Antibody on Protein A Column: Approximately 75 mg ofammonium sulfate precipitated protein (derived from 8 ml ascites) wascontinuously agitated by rocking overnight at 4° C. with 5 ml protein ASepharose beads to allow IgG to bind. The beads were poured into the 10ml column noted above and washed thoroughly with approximately 50 ml PBSpH 8.0 until protein was no longer detected in the eluate, as monitoredwith an ISCO UA-5 Absorbance Detector at 280 nm. The MHS-10 IgG1 boundto the beads was then eluted with approximately 100 ml PBS, pH 5.5,pumped at a flow rate of 0.6 ml/min, until protein was no longerdetected in the eluate. The column was then cleared of any remainingbound material, including other isotypes of antibody, with 0.01Mcitrate, 0.15M NaCl, pH 3.0. Finally, the column was re-equilabrated andstored with PBS pH 8.0 and 0.2% sodium azide at 4° C.

Protein Determination: The amount of protein in each fraction as well asthat of the starting material was determined using the Micro BCA method(Pierce Chemical Co., Rockford, Ill.). This method can measure proteinconcentrations in the range of 1-20 ug/ml.

Affinity Column Preparation: Cyanogen Bromide activated Sepharose 4 B(Sigma Chemical Co., St. Louis, Mo.) was used as the immobilizing phasefor the purified MHS-10 antibody. Pharmacia Fine Chemicals AB, AffinityChromatography: Principles and Methods, 1979, Uppsala Sweden, pp 12-18.!Three grams of the dry beads were swollen to a column of 10 ml in 1 mMHCL for 15 minutes and then washed in 200 ml of the same. The beads werewashed with coupling buffer (0.1M NaHCO₃, pH 8.3, with 0.5M NaCl) andimmediately transferred to 15 ml of a solution of 32 mg purified MHS-10IgG in coupling buffer. The mixture was agitated by rocking overnight at4° C. to allow antibody to bind. The beads were then washed with 100 mlcoupling buffer. Unreacted active sites on the beads were blocked byincubating with 0.1M Tris, 0.1M glycine, and 0.5M NaCl, pH 8.3, for 3hours at room temperature. The beads were poured into the column, a 12ml disposable syringe fitted with a teflon support and 3-way stopcock,and washed again with coupling buffer. They were then washedsequentially with acetate buffer (0.1M acetate, 0.5M NaCl, pH 4.0),coupling buffer, and then acetate buffer, and finally were equilibratedwith 0.1M Hepes pH 8.0 with 0.2% sodium azide for storage. A BCA proteinassay on the material which did not bind to the beads indicated that 2mg of the original 32 mg of MHS-10 IgG did not bind.

Sperm Preparation: Ejaculates from 10-12 donors were allowed to liquefyfor one hour at room temperature, and then were washed twice bycentrifugation at 400×G in Ham's F10 medium buffered with 0.1M Hepes, pH7.4. The washed pellets were stored frozen at -20° C. until use. Pelletswere thawed, dounce homogenized in 5 ml 0.1M Hepes pH 8.0, andmicrocentrifuged at 13,000×G, and the supernatant was filtered through a0.45 um Millipore membrane filter.

Affinity Chromatography: The sperm extract was pumped continuously overthe monoclonal antibody affinity column at 1.3 ml/min. at 4° C. to allowthe SP-10 to bind. Material that did not bind was washed from the columnwith 0.1M Hepes, pH 8.0, and the protein present in the eluate wasmonitored with an ISCO UA-5 Absorbance Detector at 280 nm wavelength.After complete removal of unbound material, bound SP-10 was eluted with0.1M glycine, 0.15M NaCl, pH 2.4. Fractions of eluting antigen weremonitored by UV absorbance, SP-10 fractions were pooled to a volume of30 ml which was then concentrated to 0.7 ml using an Amicon Centricon 10membrane filtration concentrator with 10,000 MW cutoff.

Reverse Phase HPLC: The SP-10 fraction from the affinity column wasfurther purified by reverse phase HPLC on a Brownlee 7mm×250 mm semiprepcolumn packed with Aquapore C-8, 7 um silica, 300 A pore size, (No.C03-257). A Gilson HPLC with 704 system manager and 620 data mastersystem was used. The mobile phase gradient was 0-80% solvent B over 50minutes. Solvent A was 0.1% TFA (trifluoroacetic acid, Baker Chemicals)in distilled water, and solvent B was 0.1% TFA in HPLC grade 2-propanol(Baker). Eluted protein was monitored with a model 116 UV detector at214 nm wavelength, and separate peaks were collected manually. Fractionsfrom the HPLC were frozen and then dried with a Savant Speed-Vac.

Polyacrylamide Gel Electrophoresis: The dried fractions were dissolvedin 50 ul sample buffer Laemmli, Nature (Lond) (1970) 227:680-685! andelectrophoresed on a 1.5 mm, 10% polyacrylamide gel in a Bio-Rad Protean16 cm apparatus at 45 mA for approximately two hours.

Electroblotting onto Polyvinylidene Difluoride (PVDF) Membranes: Theprotein bands were electroblotted onto PVDF membranes according to themethod of Matsudaira, J Biol Chem 262:10035-10038 (1987). The gels weresoaked for 5 minutes in transfer buffer, 0.01M CAPS (3-cyclohexylamino!-1-propanesulfonic acid), 10% methanol, pH 11.0, toreduce the amount of Tris, SDS and glycine. ProBlott brand PVDF (AppliedBiosystems, Inc., Foster City, Calif.) was found to bind significantlymore SP-10 protein than other brands tested (data not shown). A sheet ofProBlott was wet in methanol and soaked in transfer buffer, and thensandwiched with the gel between sheets of Whatman 3 mm chromatographypaper in a transblotting apparatus. Electroblotting proceeded for 40minutes at 0.5 amp in transfer buffer. The PVDF membrane was washed indistilled water for 5 minutes, stained with 0.1% Coomassie Blue R-250 in50% methanol for 5 minutes, and destained in 50% methanol, 10% aceticacid for 10 minutes. After a final wash in distilled water, the PVDF wasallowed to dry and stored at -20° C.

Amino Acid Sequencing: Amino acid sequencing was performed in theUniversity of Virginia Protein and Nucleic Acid Sequencing Facility. TheN-terminal amino acid sequence was determined using an AppliedBiosystems 470A Gas Phase Protein Sequenator with on-line 120A PTHanalyzer. The manufacturer's protocols were modified as describedSpeicher, Techniques in Protein Chemistry, Hughi T. E., ed., AcadmicPress, pp 24-35 (1989)! to improve sequencing efficiency with samples onPVDF. Dried protein bands on PVDF were excised and loaded directly intothe sequencer. One cycle was performed without phenylisothiocyanate(PITC) followed by up to 15 cycles with PITC. Cleavage of the N-terminalamino acids was accomplished via gas phase trifluoracetic anhydrideresulting in the formation of anilinothiazolinone (ATZ) derivatives. ATZamino acids were converted to PTH amino acids by heating in 25%trifluoroacetic acid for 9 minutes at 55° C. The PTH derivatives or amixture of PTH standards were analyzed on an Online Applied Biosystems120A PTH Analyzer with an Applied Biosystems C18 reverse phase column.PTH derivatives were eluted with a gradient of acetonitrile in 5%tetrahydrofuran and 70 mm sodium acetate, pH 3.9 and detected at 269 nm.Because of the relatively small amount of protein that was sequenced andbackground peptides, some amino acids were incorrectly identified whencompared to the sequence translated from DNA.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products and processesof the present invention. Thus, it is intended that the presentinvention covers such modifications and variations, provided they comewithin the scope of the appended claims and their equivalents.

REFERENCES

1) Kurth B. E., Klotz K., Flickinger C. J., Herr J. C. Localization ofsperm antigen SP-10 during the six stages of the cycle of theseminiferous epithelium in man. Biol Reprod 1991; 44:814-821.

2) Herr J. C., Flickinger C. J., Homyk M., Klotz K., John E. Biochemicaland morphological characterization of the intraacrosomal antigen SP-10from human sperm. Biol Reprod 1990; 42:181-193.

3) Anderson D. J., Johnson P. M., Jones W. R., Griffen P. D. Monoclonalantibodies to human trophoblast and sperm antigens: report of twoWHO-sponsored workshops, Jun. 30, 1986-Toronto, Canada. J. ReprodImmunol 1987; 10:231-257.

4) Wright R. M., John E., Klotz K., Flickinger C. J., Herr J. C. Cloningand sequencing of cDNAs coding for the human intra-acrosomal antigenSP-10. Biol Reprod 1990; 42:693-701.

5) Herr J. C., Wright R. M., John E., Foster J., Kays T., Flickinger C.J. Identification of human acrosomal antigen SP-10 in primates and pigs.Biol Reprod 1990; 42:377-382.

6) Dubova-Mihailova M., Mollova M. Ivanova M., Kehayov I., Kyurkchiev S.Identification and characterization of human acrosomal antigen definedby a monoclonal antibody with blocking effect on in vitro fertilization.J Reprod Immunol 1991; 19:251-268.

7) Herr J. C., Wright P. M., John E., Klotz K., Homyk M., Foster J.,Flickinger C. J. Monoclonal antibody MHS-10 and its cognateintra-acrosomal antigen SP-10. In: Alexander N.J., Griffin D., SpielerJ. M., Waites G. M. H. (eds.), Gamete Interaction--Prospects forImmunocontraception. New York: Wiley-Liss, Inc.; 1990:13-36.

8) Sambrook J., Fritsch E. F., Maniatis T. Extraction, purification, andanalysis of MRNA from eukaryotic cells. In: Nolan C. (ed.), Molecularcloning: A laboratory manual. 2nd ed., New York: Cold Spring HarborLaboratory PRess; 19889: 7.18-7.22.

9) Ausubel F. M., Brent R, Kingston R. E., Moore D. D., Seidman J. G.,Smith Ja., Struhl K. (eds.). Preparation and analysis of RNa. In:Current protocols in molecular biology. New York: Greene PublishingAssoc. and Wiley-Interscience; 1989: 4.5.1-4.5.2.

10) Kraft R., Tardiff J., Krauter K. S., Leinwand La. Using mini-prepplasmid DNA for sequencing double stranded templates with Sequenase.1988; 6:544-546.

11) Kozac M. Compilation and analysis of sequences upstream from thetranslational start site in eukaryotic mRNAs. Nucl Acids Res 1984;12:857-873.

12) Wright R. M., Suri A., Kornreich H. B., Herr J. C. 1992. The Cloningand Characterization of the Gene Coding for the Human Acrosomal ProteinSP-10. (Manuscript in preparation.)

13) Pless D. D., Lennarz W. J. Enzymatic conversion of proteins toglycoproteins. Proc Natl Acad Sci USA 1977; 74:134-138.

14) Hart G. W., Brew K., Grant G. A., Bradshaw R. A., Lennarz W. J.Primary structural requirements for the enzymatic formation of theN-glycosidic bond in glycoprotein studies wit natural and syntheticpeptides. J Biol Chem 1979;

254:9747-9753.

                                      TABLE I                                     __________________________________________________________________________    Segregation of DNA Probe SP-10 with Human Chromosomes in EcoRI digested       Human-Mouse Cell Hybrid DNA                                                   __________________________________________________________________________                                                               Trans-                          Human Chromosomes                             loca-              HYBRID                                                                             DNA#                                                                              SP-10                                                                             1 2 3 4 5 6 7 8 9 10                                                                              11                                                                              12                                                                              13                                                                              14                                                                              15                                                                              16                                                                              17                                                                              18                                                                              19                                                                              20                                                                              21                                                                              22                                                                              X tions              __________________________________________________________________________    ATR-13                                                                              48 -   + + + + + + + + - + - + + + + +       +                                                                             +                                                                             +                                                                             - - - t 5/X                DUA- 233 -   - + - - - - + + - - - - + + - -       +                                                                             -                                                                             -                                                                             - - - -                    3BSAGA                                                                        DUA- 197 +   - - + - + - - - - - + - - + - -       +                                                                             +                                                                             -                                                                             - + - -                    5BSAGA                                                                        DUA-6                                                                              859 -   - + - + + - - - - - - - - - + -       -                                                                             +                                                                             +                                                                             - - - +                    DUM-13                                                                             186 +   + + + - + + + - - + + + - + t +       +                                                                             +                                                                             +                                                                             + + + t X/15,                                                                         15/X               JSR-2                                                                              389 -   - - + + - - + - - - - - + + - -       -                                                                             -                                                                             -                                                                             - - - +                    JSR-14                                                                             402 -   - + + + + + - - - - - + + - - -       +                                                                             -                                                                             -                                                                             + + - +                    JSR-17S                                                                             44 +   + + + - + - t + + + + + + + + +       +                                                                             +                                                                             -                                                                             + + + - 7/9                JWR-22H                                                                            653 +   t t + + - + - + - + + + + + + -       +                                                                             +                                                                             -                                                                             + + - - 2/1                JWR-26C                                                                            187 +   t + + + + + + - + + + + - + + +       +                                                                             +                                                                             -                                                                             + + - + 1/2                NSL-16                                                                             192 -   - - + + + - + + t + - + - + + +       +                                                                             +                                                                             -                                                                             + + - - 17/9               REW-11                                                                              42 +   - - - + - - + - - - + + + - - +       -                                                                             -                                                                             -                                                                             + + + +                    REX- 184 -   - - + - - - - - - + - - - + + -       -                                                                             +                                                                             -                                                                             - - - -                    11BSAgB                                                                       REX- 254 -   - - + - - - - - - + - - - + - -       -                                                                             +                                                                             -                                                                             - - t t 22/X               11BSHF                                                                        RSR-3                                                                              1162                                                                              +   - - - + - - + - - + + - - + - +       +                                                                             -                                                                             -                                                                             - + - +                    SIR-8                                                                              673 +   + + + + + - + + + + + + + + + +       +                                                                             +                                                                             +                                                                             - + + +                    SIR-11                                                                             390 -   - - - - - - + - - - - - + - - -       -                                                                             -                                                                             -                                                                             - + + +                    TSL-1                                                                              643 +   - + + + - - - - - + + - + - - +       +                                                                             +                                                                             -                                                                             + + - -                    TSL-2                                                                              644 -   - + t - + + - + - + - + - + + -       t                                                                             +                                                                             -                                                                             + + - + 17/3               VTL-6                                                                              395 +   - + - - - + + + - + + - - - + -       +                                                                             -                                                                             +                                                                             + + + -                    WIL-1                                                                               20 -   - - - - - - - + - - - + - + - -       +                                                                             +                                                                             -                                                                             - + - +                    WIL-2                                                                               12 -   - - - - - - - + - - - + - - + -       +                                                                             -                                                                             -                                                                             - + - +                    WIL-5                                                                               9  -   - - - + - - - + - + - - - - - -       +                                                                             +                                                                             -                                                                             - + - +                    WIL-7                                                                               13 +   - + + - + + - + - + + - + + - -       +                                                                             +                                                                             -                                                                             - + - +                    WIL-14                                                                             347 -   + - + - + - + + - + - + - + + -       +                                                                             -                                                                             -                                                                             - - - +                    WIL-15                                                                              25 +   - + + + - + + - - + + + + + + -       +                                                                             +                                                                             -                                                                             + + - +                    W12  559 +   - + - - + - - - - - + - - + - -       +                                                                             -                                                                             +                                                                             + + + - 11p-               XOL-6                                                                              534 +   t - - - + + + - - + + + - + - -       +                                                                             -                                                                             +                                                                             + - + t 1/X                XOL-9                                                                              554 -   t + + + - + - - - - - + - - + -       +                                                                             +                                                                             +                                                                             - + + + X/1                XOL-21                                                                             1107                                                                              +   - - + - - - t + + + + + - + - -       +                                                                             +                                                                             -                                                                             + - - + ISO7p              XTR-  57 -   - - t - - - - - + t - + - - - -       -                                                                             -                                                                             -                                                                             + + - t 3/X,               3BSAgB                                                     10q-               EXR-  64 -   + + + + + + + + + + t + + + + -       +                                                                             +                                                                             +                                                                             + + + + X/11               5CSAz                                                                         EXR- 952 -   + + + + + + + + - + t + + + + -       +                                                                             +                                                                             +                                                                             + + + + X/11               5CSAZ                                                                         XER-7                                                                              640 +   + + + + + + + + + + t + + + + -       -                                                                             +                                                                             +                                                                             - + - + 11/X               XER-7                                                                              961 +   + + + + + + + + + + t + + + + -       -                                                                             +                                                                             +                                                                             - - - + 11/X               Chromosome   1 2 3 4 5 6 7 8 9 10                                                                              11                                                                              12                                                                              13                                                                              14                                                                              15                                                                              16                                                                              17                                                                              18                                                                              19                                                                              20                                                                              21                                                                              22                                                                              X                    __________________________________________________________________________    Concordant # of Hybrids                                                                    18                                                                              21                                                                              18                                                                              18                                                                              20                                                                              21                                                                              20                                                                              16                                                                              21                                                                              22                                                                              31                                                                              18                                                                              20                                                                              21                                                                              16                                                                              23                                                                              20                                                                              19                                                                              20                                                                              23                                                                              21                                                                              20                                                                              12                   Discordant # of Hybrids                                                                    13                                                                              13                                                                              15                                                                              17                                                                              15                                                                              14                                                                              13                                                                              19                                                                              13                                                                              12                                                                               0                                                                              17                                                                              15                                                                              14                                                                              18                                                                              12                                                                              14                                                                              16      15                                                                            12                                                                            14                                                                            14                                                                              18                   % Discordancy                                                                              42                                                                              38                                                                              45                                                                              49                                                                              43                                                                              40                                                                              39                                                                              54                                                                              38                                                                              35                                                                               0                                                                              49                                                                              43                                                                              40                                                                              53                                                                              34                                                                              41                                                                              46      43                                                                            34                                                                            40                                                                            41                                                                              60                   __________________________________________________________________________

We claim:
 1. A monoclonal antibody specific for an antigen of FIGS 11Aand 11B, and said antigen being testis specific and persistentthroughout spermiogenesis, and wherein said antibody has the epitopicbinding characteristics of the monoclonal antibody expressed by cellline ATCC HB
 10039. 2. The monoclonal antibody of claim 1, wherein saidantibody is complexed with a reporter moiety selected from the groupconsisting of an enzyme, a dye and a radioactive label.
 3. Themonoclonal antibody of claim 1, wherein said monoclonal antibody isaffixed to a solid support.
 4. The monoclonal antibody of claim 3,wherein said solid support is an immunoaffinity bead.
 5. The monoclonalantibody of claim 2, wherein the monoclonal antibody is the antibodyexpressed by cell line ATCC HB
 10039. 6. An immunochemical assay fordetermining the fertility or infertility of a male human individual,comprising:obtaining a semen sample from said individual, combining saidsemen sample with a monoclonal antibody of claim 1, under conditionswhich permit said monoclonal antibody to bind to an antigen present inthe sample, said antigen being testis specific and persistent throughoutspermiogensis, and observing said semen sample combined with saidantibody to determine the presence of said binding, wherein theoccurrence of said binding is indicative of fertility in saidindividual, and the absence of said binding is indicative of infertilityin said individual.
 7. The assay of claim 6, wherein said monoclonalantibody is the monoclonal antibody expressed by cell line ATCC HB10039.
 8. A method of inhibiting fertilization of ova in a femalemammal, comprising immunizing said female with an amount of an antigensufficient to induce the production of antibodies thereto in saidfemale, wherein said antigen of FIGS. 11A and 11B, is an intra-acrosomalmammalian sperm antigen that remains associated with said mammaliansperm after the acrosome reaction, wherein said antigen is bound bymonoclonal antibodies produced by cell line ATCC HB 10039 and whereinsaid female is sufficiently immunized so as to produce afertilization-inhibiting amount of circulating antibody.
 9. Acontraceptive composition comprising an amount of the antibody of claim1 in a carrier compatible with a vaginal environment, wherein saidamount is effective to promote binding of said antibody to substantiallyall sperm present in a ejaculate of a mature male of the species of saidfemale.
 10. A method of inhibiting fertilization of a mammalian female,comprising of applying the contraceptive composition of claim 9 to thevagina of said female in advance of intercourse.